Compact, low dispersion, and low aberration adaptive optics scanning system and method

ABSTRACT

An adaptive optics scanning system and method using a beam projection module with four or more axes of motion that can project and control the position and angle of a beam of light to or from an adaptive optics element. The adaptive optics scanning system is compact in size, overcoming the challenges of a traditional lens and mirror based pupil relay design. The adaptive optics scanning system has little to no dispersion, chromatic aberration, and off-axis aberration for improved optical performance. The system and methods for calibrating and optimizing the system are described. A modular adaptive optics unit that scans and interfaces an adaptive optics element is described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-provisional applicationSer. No. 14/052,268 filed on Oct. 11, 2013, which claims the benefit ofU.S. Provisional Application No. 61/713,478 filed on Oct. 12, 2012. Thecontents of U.S. Non-provisional application Ser. No. 14/052,268 andU.S. Provisional Application No. 61/713,478 are herein incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to the field of adaptive optics beamscanning.

BACKGROUND

Most optical systems are designed with consideration of the opticalaberrations internal to the system only. Careful selection of opticalsurface geometry combined with precise fabrication, careful assembly,and inclusion of a select few adjustable parameters (e.g. focus, zoom,or spherical aberration correction) allow the optical system to achievea specified nominal level of performance. However, if a source ofoptical aberration exists outside of the optical system and theaberrations are unknown and possibly changing with time, the performanceof the optical system can be significantly degraded. A select fewexamples of beam scanning imaging systems and sources of aberration areshown in FIG. 1 and FIG. 2, respectively. Adaptive optics (AO) providesa means to reduce the wavefront distortions caused by the source ofaberration to achieve improved performance. In most AO systems, awavefront correcting device (often a deformable mirror or liquid crystalspatial light modulator) contains several to thousands of individuallyaddressable actuators or cells (pixels) to affect the wavefront, asshown in FIG. 3. Undesirable distortions to the wavefront can becorrected or a more preferable wavefront shape generated with thewavefront correcting device integrated in the optical system. Adaptiveoptics has been applied to correct for dynamic atmospheric aberrationfor telescope viewing, to correct for aberrations in the human andanimal eye for retinal imaging, to correct for sample inducedaberrations for microscopic imaging, to correct for sample inducedaberrations in laser material processing, to correct for atmosphericaberrations for line of sight optical communications, and otherapplications where wavefront correction is desirable. The benefits ofadaptive optics are generally improved resolution and signal strength inviewing or imaging applications, tighter focus and higher power densityin beam projection applications, or improved communication rates in datatransmission applications.

A paper, “The Possibility of Compensating Astronomical Seeing”, H. W.Babcock, Publications of the Astronomical Society of the Pacific, Vol.65, No. 386, p. 229 (1953) first introduced the adaptive optics conceptfor astronomical viewing with earth based telescopes. The vast majorityof adaptive optics systems to date have used the basic AO frameworkproposed in Babcock's paper with the system containing a wavefrontsensor 410, an adaptive optics element 420, and a feedback controlsystem 430 that takes input from the wavefront sensor and generatescontrol signals to drive the adaptive optics element to a preferredwavefront correction shape, as shown in FIG. 4(A). The wavefront sensorcould be of a Shack-Hartmann, pyramid, or other wavefront sensingdesign. An alternate and more recent implementation of AO does not use awavefront sensor, but instead uses information about the quality of themeasured signal as obtained by the image sensor 440 as the input to anoptimization algorithm running on an optimization system 450 as part ofthe process to generate wavefront corrections for the adaptive opticselement 460 for improved performance, as shown in FIG. 4(B).Implementing AO in this manner when the wavefront correction is notknown a priori and without a dedicated wavefront sensor is commonlyreferred to as sensorless AO. A third variation of AO uses stored orcalculated control signals applied to the adaptive optics element 470 byan open loop control system 480, referred to as open loop AO, as shownin FIG. 4(C).

AO System Aberration Challenges Taught by AO-SLO Examples

The historical challenges of managing system aberrations are describedin the context of adaptive optics scanning laser ophthalmoscopes(AO-SLO). It has been long known that the peripheral cornea andcrystalline lens in the human eye introduce wavefront distortions thatdegrade resolution at large pupil diameters. A paper, “Optical qualityof the human eye” by F. W. Campbell and R. W. Gubisch, Journal ofPhysiology, Vol. 186, no. 3, pp. 558-578 (1966), finds that a pupildiameter of 2.4 mm yields the highest optical resolution usinglinespread analysis. Similar findings in a more recent paper, “Optimalpupil size in the human eye for axial resolution” by W. J. Donnelly IIIand A. Roorda, JOSA. Vol. 20, Issue 11, pp. 2010-2015 (2003), indicatethat a pupil size of 2.46 mm provides the best lateral resolution and4.6 mm provides the best axial resolution for traditional (non-AO)scanning laser ophthalmoscope (SLO) imaging. The aberration associatedwith larger pupil sizes dominates and degrades resolution to a greaterextent than the improvement of resolution expected with the increasingnumerical aperture and associated improved diffraction limit. Anadaptive optics element can correct the peripheral cornea andcrystalline lens aberrations to allow larger pupil diameters to be usedat or near the diffraction limit to achieve significantly improvedresolution and imaging performance.

A paper, “Active optical depth resolution improvement of the lasertomographic scanner” by A. Dreher, J. Bille, and R. Weinreb, Appl. Opt.28, 804-808 (1989) teaches using a deformable mirror in an open loopmanner to correct for aberrations in the human eye at a pupil diameterof 6 mm to achieve a two-fold increase in depth resolution in a lasertomographic scanner. Further, the same paper teaches using an afocal 4farrangement of lenses in a relay configuration to image the activesurface of the deformable mirror to the entrance pupil of the eye. Anadditional afocal 4f arrangement of lenses images the scan pupil of agalvanometer (galvo) scanner to the active surface of the deformablemirror. This basic arrangement and use of multiple 4f relays between theeye, AO element, and scanners has become the standard for nearly all AOsystems that perform laser scanning ophthalmic imaging, although theordering of pupil planes and specific optical components used in the 4frelay can differ. If an additional galvanometer is used to perform 2Dscanning, an associated additional 4f relay is used for proper pupilconjugation to the other scanner, adaptive optics element, and pupilplanes. The design of the 4f pupil relay has been challenging becauseoff-axis aberrations in the imaging system itself can introducesignificant wavefront distortions. The problem is exacerbated becauseaberrations compound as multiple 4f relays are cascaded in series.

Early point scanning adaptive optics imaging systems used sphericalmirrors in off-axis configurations to perform the 4f pupil relay andprimarily concentrated on optimizing the image plane performance, as isdescribed in a paper, “Adaptive optics scanning laser ophthalmoscopy” byA. Roorda, F. Romero-Borja, W. Donnelly, III, et al, A. Roorda, F.Romero-Borja, W. Donnelly, III Opt. Express 10, 405-412 (2002) and arelated U.S. Pat. No. 6,890,076 B2. However, the in-plane configurationof pupil relays used in this paper and patent is known today to generateconsiderable residual astigmatism aberration which degrades imagingperformance.

A paper “Large-field-of-view, modular, stabilized, adaptive-optics-basedscanning laser ophthalmoscope” by S. Burns, R. Tumbar, A. Elsner et al,J. Opt. Soc. Am. A 24, pp. 1313-1326 (2007), teaches that even withsmall off-axis beam angles on the spherical mirrors in the 4f pupilrelay, off-axis astigmatism accumulates with multiple sequential mirrorreflections in the system. The paper teaches that designing the opticssuch that the second pupil relay is constructed out-of-the-planecompared with the first pupil relay, astigmatism can be partiallycancelled. A paper, “First-order design of off-axis reflectiveophthalmic adaptive optics systems using afocal telescopes” by A.Gómez-Vieyra, A. Dubra, D. Malacara-Hernindez, and D. Williams, Opt.Express 17, pp. 18906-18919 (2009), further investigates off-axisaberrations and develops associated theory to optimize imagingperformance in the both the retinal (imaging) and pupil planes by alsousing an out-of-plane relay configuration. A follow up paper, “Geometrictheory of wavefront aberrations in an off-axis spherical mirror” by A.Gomez-Vieyra and D. Malacara-Hernández, Appl. Opt. 50, pp. 66-73 (2011),extends the aberration theory of pupil relays to higher orders and isused as the basis for an improved ophthalmic AO imaging system describedin a paper, “Reflective afocal broadband adaptive optics scanningophthalmoscope” by A. Dubra and Y. Sulai, Biomed. Opt. Express 2, pp.1757-1768 (2011).

Indeed, the importance of minimizing aberration, and particularlyastigmatism, as well as simultaneously minimizing both the aberrationsin the imaging planes and the pupil planes was demonstrated by twogroups independently publishing images of the elusive rod mosaic in thepapers, “Noninvasive imaging of the human rod photoreceptor mosaic usinga confocal adaptive optics scanning ophthalmoscope” by A. Dubra, Y.Sulai, J. Norris, R. Cooper, A. Dubis, D. Williams, and J. Carroll,Biomed. Opt. Express 2, pp. 1864-1876 (2011) and “Observation of coneand rod photoreceptors in normal subjects and patients using a newgeneration adaptive optics scanning laser ophthalmoscope” by D. Merino,J. Duncan, P. Tiruveedhula, and A. Roorda Biomed. Opt. Express 2, pp.2189-2201 (2011). This second paper also teaches that in addition tointroducing scan position dependent wavefront aberrations in both theimage and pupil planes, beam wandering also occurs in spherical mirrorbased 4f pupil relay systems. Beam wandering can be improved with theout-of-plane relay configuration.

Over the course of over a decade, AO based SLO imaging has advancedconsiderably from systems that could only resolve the relatively largeperipheral cone mosaic to being able to resolve the very small rodmosaic in the retina. Paying close attention to the details of theaberrations and quality of pupil relay has been a major contributor tothe ever improving imaging performance. However, the resulting size ofthese new optimized AO imaging systems is quite large due to the longfocal lengths of the spherical mirror components used in the highlyoptimized designs. For example, in the previously mentioned optimizeddesigns, the afocal telescope is over 1.5 meters in length (Dubra, 2011)and 0.4 meters in length (Merino, 2011) because long focal lengthmirrors are used to reduce off-axis aberration. The large size of thespherical mirror based AO systems is compounded by the need to cascademultiple afocal relays in the AO system, each of considerable length ofits own.

Positively powered mirrors and reflective surfaces have been mostcommonly used in AO-SLO systems because the small back reflections fromglass or lens surfaces are significant and can interfere withmeasurement of the small levels of light returning from the retina.Glass surface back reflections can also generate stray light artifactsand ghost images that degrade wavefront measurement with a wavefrontsensor. For these reasons, mirrors have been preferred over lenses andhave been used almost exclusively in high performance AO-SLO systems, asdescribed in the before mentioned paper (Gómez-Vieyra, 2009).

A paper, “Lens based adaptive optics scanning laser ophthalmoscope” byF. Felberer, J. Kroisamer, C. Hitzenberger, and M. Pircher, Opt. Express20, 17297-17310 (2012), teaches that an all lens based implementation ofthe multiple afocal pupil relays used in an AO-SLO system can achieve acomparable level of aberration as the more complicated out-of-planespherical mirror based configuration. The lengths of the afocal pupilrelays are on the order of 0.5 meters. The problem of backreflectionsfrom the glass surfaces interfering with the wavefront measurement isaddressed by introducing a polarization beam splitter and polarizer infront of the wavefront sensor and a quarter waveplate in front of theeye such that light reflected from glass surfaces is rejected, but lightreflected from the eye is passed through to the wavefront sensor. Theproblem of backreflections from lens and glass surfaces interfering withthe image detection and formation is not addressed. The paper showsresults of the rod mosaic, although the quality of the image does notlook as good as the images obtained with the all mirror basedout-of-plane configuration of the before mentioned Dubra 2011 paper.

The discussion so far has focused on AO-SLO because this technology isone of the most well documented and carefully analyzed of the adaptiveoptics systems. Other AO systems using different imaging modalities ormaterial processing capability have also been demonstrated and havefaced the same off-axis aberration and size challenges, as well asadditional challenges associated with dispersions in glass elements whenshort pulsed lasers are used.

Microscope Imaging with Adaptive Optics

High performance microscope objectives achieve optimal performance whenimaging under well controlled and prescribed imaging conditions. Smallperturbations to nominal imaging conditions can result in a significantreduction of signal strength and a degradation of resolution.Detrimental perturbations to nominal imaging conditions can arise fromusing different thickness coverslips, using an oil immersion objectivein a water immersion imaging scenario, from imaging into tissue or othersamples, from imaging through sample containers, or from other sources.A paper, “Aberration correction for confocal imaging inrefractive-index-mismatched media” by M. J. Booth, M. A. A. Neil, and T.Wilson, Journal of Microscopy, Vol. 192, issue 2, (1998) analyzesspecimen and sample induced aberration and teaches the potential ofusing a deformable mirror in a confocal or two photon microscope tocorrect for aberrations occurring from deep imaging through refractiveindex mismatched media.

A paper, “Adaptive aberration correction in a two-photon microscope” byM. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S.Kawata, J. Microscopy, Vol. 200, Pt. 2, pp. 105-108 (2000), describesthe first experimental application of two photon imaging with adaptiveoptics. The adaptive optics corrector, a ferroelectric liquid crystalspatial light modulator (FLCSLM), is located before the scanningmechanism in a commercial laser scanning microscope.

A patent, U.S. Pat. No. 6,381,074 B2, teaches a laser scanningmicroscope that includes a wavefront converting element to performscanning of the focus in the optical axis (depth) direction without theneed to change the distance between the microscope objective and thespecimen. Aberration occurring during the depth scanning is canceled byusing the wavefront converting element to minimize the degradation oflight collecting performance due to the scanning in the optical axisdirection. The wavefront converting element is placed at or near aposition conjugate to the objective pupil position so that predeterminedconditions are satisfied. Further, the wavefront converting element andeach of two galvanometer mirrors in the scanning optical system to scanthe position where light is collected in a direction perpendicular tothe optical axis and further the pupil position of the objective are allplaced in conjugate or nearly conjugate relation to each other by theintervening optical systems. The scanning optical system includes apupil projection lens for placing the wavefront converting element andthe galvanometer mirror closer to the wavefront converting element inconjugate relation to each other.

A paper, “Smart microscope: an adaptive optics learning system foraberration correction in multiphoton confocal microscopy” by O. Albert,L. Sherman, G. Mourou, T. Norris, and G. Vdovin, Opt. Lett. 25, 52-54(2000) teaches using a deformable mirror to correct for off-axisaberrations in a two photon imaging system. The objective is an off-axisparabolic mirror and the intensity of a two photon sample is used tooptimize the deformable mirror shape.

A paper, “Adaptive aberration correction in a confocal microscope” by M.J. Booth, M. A. A. Neil, R. Juskaitis and T. Wilson, Proc. Nat. Acad.Sci., Vol. 99, No. 9, 30, pp. 5788-5792 (2002), describes the firstdemonstration of adaptive optics in a confocal microscope. The paperteaches using relay lenses between the deformable mirror and theobjective.

A paper, “Adaptive correction of depth-induced aberrations inmultiphoton scanning microscopy using a deformable mirror” by Sherman L,Ye J Y, Albert O, Norris T B. J Microsc. 206 (Pt 1):65-71 (2002),demonstrates using a deformable mirror as the wavefront corrector in amultiphoton scanning microscope. The paper teaches using a 4f telescopesystem to directly image the face of the DM to the entrance pupil of themicroscope objective.

A patent, U.S. Pat. No. 6,771,417 B1, teaches the use of one or morewavefront modulators in the observation beam path and/or illuminationbeam path of a microscope. The patent teaches placing the wavefrontmodulator between the tube lens and the objective. Such modulators maybe adapted to change the phase and/or the amplitude of light in such away to carry out displacement and shaping of the focus in the objectspace and correction of possible aberrations. An embodiment of theinvention allows focusing to different depths without changing thedistance from the objective to the object. The possible areas of useinclude confocal microscopy, laser-assisted microscopy, conventionallight microscopy and analytic microscopy.

A patent, U.S. Pat. No. 7,733,564 B2 (continuation patent of abovementioned U.S. Pat. No. 6,771,417 B1), includes additional claims inwhich a design change to the instrument of placing the wavefrontmodulator in a pupil plane is claimed, although the methods andmechanisms for doing so are not described.

A patent, U.S. Pat. No. 7,659,993 B2, teaches a wavefront sensing devicewithin an adaptive optics microscope architecture. An embodiment of theinvention is described for fluorescent imaging with examples ofmulti-photon and confocal microscopy. A wavefront sensor usesinterferometric techniques, called coherence gating, to isolate a depthof interest in the sample. The deformable mirror is adapted to apredetermined shape in order to form the desired wave-front of thetravelling light pulses. Specimen scanning is obtained with movement ofthe specimen holding device.

The challenges of using the before mentioned and traditional approach ofcascading multiple pupil relays in microscopy has been recognized. Apatent, U.S. Pat. No. 7,002,736 B2, teaches a scanning opticalmicroscope using a wavefront converting element to correct foraberrations. Citing Japanese patent, HEI-11-101942 4 (1999), whichteaches that it is desirable that the wavefront converting elementshould be placed at a position conjugate to the pupil, the patentemphasizes that it is difficult to implement pupil relay systems becauseof the following problems. A first problem is that a variety ofobjectives are used in microscopic observation, and the pupil positiondiffers for each objective. Therefore, when a plurality of objectivesare switched from one to another to perform observation, it is difficultto keep the pupils of the objectives in conjugate relation to thewavefront converting element at all times. Further, the wavefrontconverting element needs to be placed in conjugate relation to theposition of a laser scanning member and also to the position of theobjective pupil. Accordingly, at least two pupil relay optical systemsare required. Therefore, the apparatus becomes large in size andcomplicated unfavorably.

Adaptive optics have been used in a microscope for reasons other than tocorrect optical aberrations. A patent, U.S. Pat. No. 8,198,604 B2,teaches a system for providing enhanced background rejection in thicktissue that contains an aberrating element for introducing controllableextraneous spatial aberrations in an excitation beam path. An associatedmethod comprises the steps of acquiring two-photon excited fluorescenceof thick tissue without extraneous aberrations; introducing anextraneous aberration pattern in an excitation beam path; acquiringtwo-photon excited fluorescence of the thick tissue having theintroduced extraneous aberration pattern; and subtracting the two-photonexcited fluorescence with extraneous aberrations from the acquiredstandard two-photon excited fluorescence of the thick tissue withoutextraneous aberrations. The deformable mirror is relayed to the beamscanner, which is in turn relayed to the back aperture of the objective.The deformable mirror is located in a conjugate plane of the objectiveback aperture.

OCT Imaging with Adaptive Optics

Similar to AO-SLO, adaptive optics has been applied to Optical CoherenceTomography (OCT) for adaptive optics OCT (AO-OCT).

A patent, U.S. Pat. No. 7,364,296 B2, teaches a method of opticalimaging comprising providing a sample to be imaged, measuring andcorrecting aberrations associated with the sample using adaptive optics,and imaging the sample by optical coherence tomography.

A patent, U.S. Pat. No. 7,942,527 B2, teaches using a Badal optometerand rotating cylinders inserted in an AO-OCT system to correct largespectacle aberrations such as myopia, hyperopic and astigmatism for easeof clinical use and reduction. Similar to as implemented with AO-SLO,spherical mirrors in the telescope are rotated orthogonally(out-of-plane) to reduce aberrations and beam displacement caused by thescanners. This produces greatly reduced AO registration errors andimproved AO performance to enable high order aberration correction inpatient eyes.

A patent, U.S. Pat. No. 7,896,496 B2, teaches an object tracking systemthat can be used for AO-SLO or AO-OCT.

A patent application, WO2005060823 A1, teaches a data acquisition systemwhere measurements are made by OCT, wherein a quality of thesemeasurements is improved by arranging an active optical element in thebeam path, the system also including a wavefront sensor.

A patent application. US20120019780 A1, teaches an AO-SLO or AO-OCT.

A patent application, US20110234978 A1, teaches a multifunctionaloptical apparatus that includes a system of optical components capableof operating in a scanning laser ophthalmoscope (SLO) mode and anoptical coherence tomography (OCT) mode. Multiple scanning devices arepositioned at pupil conjugates in the system of optical components. Thesystem may include optical tracking along with one or more optionaladaptive optics.

A patent application, US20120002165 A1, teaches an invention that canimage with SLO or OCT that has multiple measuring beams and usesadaptive optics that include: a wavefront aberration detector fordetecting a wavefront aberration in a reflected or backscattered beamsgenerated when a plurality of beams are scanned on a surface, and asingle wavefront aberration corrector for correcting a wavefrontaberration in each of the plurality of beams, based on the wavefrontaberration, and the plurality of beams enter the single wavefrontaberration corrector with different incident angles and are overlappedon each other. In one embodiment, the wavefront aberration corrector isdisposed at a position at which an exit pupil of relay optics isacquired optically conjugate with the single position at which theplurality of beams intersect with each other.

A patent application, US20120044455 A1, teaches an AO-SLO or AO-OCTimaging apparatus using a deformable mirror and wavefront sensor. Pupilrelay optics are used and the patent application teaches that relaylenses are used so that the cornea, the XY scanner, and the wavefrontsensor become approximately optically conjugate with each other.

Material Processing and Object Manipulation with Adaptive Optics

Various papers have described using adaptive optics for beam shaping inmaterial processing applications, including a paper, “Beam delivery byadaptive optics for material processing applications using high-powerCO2 lasers” by Heinz Haferkamp and Dirk Seebaum, Proc. SPIE 2207, LaserMaterials Processing: Industrial and Microelectronics Applications, 156(1994), and a paper, M. Geiger, Synergy of Laser Material Processing andMetal Forming, CIRP Annals—Manufacturing Technology, Volume 43, Issue 2,pp. 563-570 (1994).

Adaptive optics have been used to correct for sample induced aberrationin material processing. A paper, “Active Aberration Correction for theWriting of Three-Dimensional Optical Memory Devices” by M. Neil, R.Juskaitis, M. Booth, T. Wilson, T. Tanaka, and S. Kawata, Appl. Opt. 41,1374-1379 (2002), teaches using an SLM to compensate for sample inducedaberrations when writing 3D optical memory devices. A paper, “Ultrafastlaser writing of homogeneous longitudinal waveguides in glasses usingdynamic wavefront correction”, C. Mauclair, A. Mermillod-Blondin, N.Huot, E. Audouard, and R. Stoian, Opt. Express 16, 5481-5492 (2008),teaches using an SLM in a laser processing system to improve the qualityof laser processing. A paper, “Adaptive optics for direct laser writingwith plasma emission aberration sensing” by A. Jesacher, G. Marshall, T.Wilson, and M. Booth, Opt. Express 18, 656-661 (2010), teaches using anSLM in a plasma emission direct laser writing system.

Adaptive optics have been used for optical manipulation. One method ofmanipulating small objects is to use optical trapping, sometimesreferred to as optical tweezers. Most methods of using optical tweezersdo not include a galvo based scanning mechanism as taught in thefollowing papers: “Adaptive optics in an optical trapping system forenhanced lateral trap stiffness at depth”, by M C Miillenbroich, NMcAlinden and A J Wright, M C Mfillenbroich et al, J. Opt. 15 075305(2013), a paper, “Holographic optical tweezers aberration correctionusing adaptive optics without a wavefront sensor” by K D. Wulff, D G.Cole, R L. Clark, R D Leonardo, J Leach, J Cooper, G Gibson, M J.Padgett, Proc. SPIE 6326, Optical Trapping and Optical MicromanipulationIII, 63262Y (2006), and a thesis, “Design and characterization of anoptical tweezers system with adaptive optic control” by S. Bowman(2009).

More advanced optical trapping setups include scanning and/or beamsplitting capability, such as a paper, “Combined holographic-mechanicaloptical tweezers: Construction, optimization, and calibration”, byRichard D. L. Hanes, Matthew C. Jenkins, and Stefan U. Egelhaaf, Rev.Sci. Instrum. 80, 083703 (2009). In this paper, the SLM is placed nearthe objective and not explicitly conjugated to the aperture. The SLMallows multiple traps to be formed such that the galvos can do coarsesteering of the beam and the SLM can perform beam splitting to generatemultiple traps and fine steering of the beam. The deformable mirror usedin the apparatus is calibrated by optimizing oscillatory drag force on atrapped object.

SUMMARY

An embodiment of the present invention is an adaptive optics scanningsystem and methods for its calibration and operation. The unique designof the adaptive optics scanning system of an embodiment of the presentinvention overcomes limitations in prior art related to large size,dispersion, chromatic aberration, and off-axis aberration. An embodimentof the present invention enables a reduction in size by replacing staticoptical elements used in a traditional design with active opticalelements to achieve proper beam centration with respect to the adaptiveoptics component. An embodiment of the present invention eliminates theneed for the 4f relays between the scanning mirrors, while at the sametime increases instrument performance, flexibility and capability. Anembodiment of the present invention overcomes the challenges of off-axisaberration associated with the traditional lens based and concave mirrorbased pupil relay configurations used in most adaptive optics systems byusing only flat or nearly flat reflective surfaces. An embodiment of thepresent invention overcomes the detrimental effects of dispersion andchromatic aberration associated with lens based designs by using onlyreflective mirrors. An embodiment of the present invention enablesimproved adaptive optics performance in a small form factor. Anembodiment of the present invention enables programmable flexibility foraccommodating different sample delivery optics. Moreover, an embodimentof the present invention is compatible with a wide range of imagingmodalities, processing methods, and characterization methods used inbiological, medical, industrial imaging and inspection. The possibleareas of use include medical imaging, biological imaging, industrialinspection, material processing, material inspection, subsurfaceimaging, surface profiling, distance ranging and measurement, fluid flowcharacterization and analysis, and investigation and characterization ofmaterial polarization properties.

One embodiment provides an adaptive optics scanning system including: anemission source for generating light, the light being directed throughthe adaptive optics scanning system to a sample; one or more adaptiveoptics element(s), the adaptive optics element(s) affecting thewavefront, affecting the intensity, or affecting both the wavefront andintensity of the light; a beam projection module, the beam projectionmodule operating with four or more axes of motion and controlling anangle and position of the light to preferentially interface the adaptiveoptics element by creating or accommodating a beam pivot point at ornear the adaptive optics element(s) while scanning the light across thesample; a controller for controlling motion trajectories of the axes inthe beam projection module; sample delivery optics, the sample deliveryoptics appropriately conditioning and directing the light to the sample;one or more detector(s), the detector(s) measuring light from thesample.

One embodiment provides a modular adaptive optics unit including: one ormore entrance ports, the entrance ports allowing one or more opticalbeams to enter the modular adaptive optics unit; one or more outputports, the output ports being located along one or more beam paths atwhich the optical beam may transit or be terminated; one or moreadaptive optics element(s), the adaptive optics element(s) affecting thewavefront, affecting the intensity, or affecting both the wavefront andintensity of the light beam; a set of beam steering elements, the beamsteering elements creating four or more axes of motion that affect anangle of, or the transverse position of, the propagation path of thelight to preferentially create at least one effective rotation pointabout which the light beam is pivoted; a means for controlling thetrajectories of the beam steering elements to direct the light beamalong preferential paths.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a collection of diagrams showing several of many exampleoptical systems and imaging modalities that can use an embodiment of thepresent invention.

FIG. 2 is a collection of diagrams showing several of many possiblesources of aberration that can be corrected using an embodiment of thepresent invention.

FIG. 3 is a collection of diagrams showing several of many possibleadaptive optics technologies that can be used in an embodiment of in thepresent invention.

FIG. 4 is a collection of block diagrams showing adaptive optics controlmethods.

FIG. 5 is a collection of diagrams showing pupil relay implementationsthat are used in optical systems with additional diagrams showing howthe relative order of optical beam steering and adaptive opticscomponents can be varied.

FIG. 6 is a collection of diagrams showing pupil relay systems with twoseparate single-axis scan mirrors or a single two-axes scan mirror.

FIG. 7 is a collection of block diagrams showing possible subsystemlayouts of an embodiment of the present invention in which the detectoris located in different positions.

FIG. 8 is a collection of diagrams showing possible characteristics ofthe emission source that is used in an embodiment of the presentinvention.

FIG. 9 is a set of diagrams showing how rotating mirrors can relay orcontrol the position of a plane of stationary beam intensity, as desiredfor an embodiment of the present invention.

FIG. 10 is a diagram showing how two pairs of rotating mirrors can bealigned to relay or control the position of a plane of stationary beamintensity in two directions.

FIG. 11 is a pair of diagrams showing views of a two directional beamsteering system that can relay or control the position of a plane ofstationary beam intensity, as viewed from the x and y axis.

FIG. 12 is a collection of diagrams showing different possibleimplementations of systems that can relay or control the position of aplane of stationary beam intensity, including systems composed of faststeering mirrors (FSMs), rotational mirrors, and translational mirrors.

FIG. 13 is a collection of diagrams showing an example implementation ofthe beam projection module of the current invention with the beam pathshown and with input and output beams indicated.

FIG. 14 is a set of solid model renderings that show the elements andbeam paths in an example implementation of the beam projection module ofan embodiment of the present invention.

FIG. 15 is a collection of drawings indicating placement and orientationof beam steering mirrors in the beam projection module of an embodimentof the present invention.

FIG. 16 is a set of block diagrams showing a controller of an embodimentof the present invention.

FIG. 17 is a set of plots showing scanning characteristics of anembodiment of the present invention.

FIG. 18 is a set of plots showing example scan patterns and scantrajectories of an embodiment of the present invention.

FIG. 19 is a ZEMAX raytrace of a simulation of an embodiment of thepresent invention showing the beam projection module integrated into alaser scanning microscope.

FIG. 20 is a collection of drawings showing lens prescriptions for aprototype embodiment of the present invention.

FIG. 21 is collection of photographs showing a prototype of anembodiment of the present invention.

FIG. 22 is a screen capture of a software program operating anembodiment of the current invention showing optimization of the adaptiveoptics element.

FIG. 23 is a pair of images obtained with a prototype of an embodimentof the present invention showing the image quality of a sample obtainedwith the deformable mirror flat and optimized that shows improvement insignal intensity and improvement in resolution in the optimizeddeformable mirror image.

FIG. 24 is a set of drawings showing OCT implementations of anembodiment of the present invention.

FIG. 25 is a set of drawings and plots showing principles of OCT imagingand optical path length change of an embodiment of the presentinvention.

FIG. 26 is a set of plots showing the effect of optical path lengthchange of an embodiment of the present invention when used for OCT.

FIG. 27 is a diagram showing an optical simulation of the human eye.

FIG. 28 is a set of drawings that show portions of an adaptive opticsimaging system where the adaptive optics element is conjugated to thepupil of a human eye.

FIG. 29 is a set of drawings that show portions of an adaptive opticsimaging system where the adaptive optics element is located outside of apupil plane in the human eye.

FIG. 30 is a set of drawings and plots comparing the optical layout andimaging performance of systems where the adaptive optics element islocated in and located outside of a pupil plane in the human eye.

FIG. 31 is a set of drawings and plots comparing a microscope imagingsystem where the adaptive optics element is located in a pupil plane andoutside of a pupil plane in a microscope system.

FIG. 32 is a collection of block diagrams showing possible systemlayouts of an embodiment of the present invention with differentordering of the adaptive optics element relative to the beam projectionmodule.

FIG. 33 is a diagram showing how adjustment of optical elements in thesample delivery optics can accommodate motion of the objective lenswhile maintaining proper conjugation of the objective lens pupil andadaptive optics element.

FIG. 34 is a collection of diagrams illustrating several of manydifferent combinations of adaptive optics technologies that can becombined in an embodiment of the present invention.

FIG. 35 is a solid model rendering of a beam alignment module that canbe used in an embodiment of the present invention to aid in alignment ofthe instrument.

FIG. 36 is a set of block diagrams showing adaptive optics controlalgorithms of an embodiment of the present invention.

FIG. 37 is an image of a screen capture showing adaptive optics controlwith a reduced basis set.

FIG. 38 is a collection of diagrams showing beam switching, a modularadaptive optics unit, and multiple beam entrance and exit ports of amodular adaptive optics unit of an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The description of illustrative embodiments according to principles ofthe present invention is intended to be read in connection with theaccompanying drawings, which are to be considered part of the entirewritten description. In the description of embodiments of the inventiondisclosed herein, any reference to direction or orientation is merelyintended for convenience of description and is not intended in any wayto limit the scope of the present invention. Relative terms such as“lower,” “upper,” “horizontal,” “vertical,” “above,” “below,” “up,”“down,” “top” and “bottom” as well as derivative thereof (e.g.,“horizontally,” “downwardly,” “upwardly,” etc.) should be construed torefer to the orientation as then described or as shown in the drawingunder discussion. These relative terms are for convenience ofdescription only and do not require that the apparatus be constructed oroperated in a particular orientation unless explicitly indicated assuch. Terms such as “attached,” “affixed,” “connected,” “coupled,”“interconnected,” and similar refer to a relationship wherein structuresare secured or attached to one another either directly or indirectlythrough intervening structures, as well as both movable or rigidattachments or relationships, unless expressly described otherwise.Moreover, the features and benefits of the invention are illustrated byreference to the exemplified embodiments. Accordingly, the inventionexpressly should not be limited to such exemplary embodimentsillustrating some possible non-limiting combination of features that mayexist alone or in other combinations of features; the scope of theinvention being defined by the claims appended hereto.

This disclosure describes the best mode or modes of practicing theinvention as presently contemplated. This description is not intended tobe understood in a limiting sense, but provides an example of theinvention presented solely for illustrative purposes by reference to theaccompanying drawings to advise one of ordinary skill in the art of theadvantages and construction of the invention. In the various views ofthe drawings, like reference characters designate like or similar parts.

Discussion of Adaptive Optics

Adaptive optics (AO) enable the correction of optical aberrations toimprove the performance of optical imaging, optical processing ofmaterials, optical profilometry, optical inspection, and other opticalcharacterization in which system, sample, or externally induced opticalaberrations degrade optical performance. Adaptive optics was originallyproposed for astronomical imaging with telescopes to compensate foraberrations introduced by the atmosphere. Using an adaptive opticselement (sometimes called a wavefront corrector) and wavefront sensor ina closed loop control system, it is possible to measure the aberrationsin the atmosphere and generate a corrective shape on the adaptive opticselement in real time to reduce the level of aberration to yield animproved image quality. Image quality generally improves in signalstrength and resolution. Adaptive optics have also been used in lasercavities, laser beam shaping, biomedical imaging, microscopy, andmaterials processing to preferentially shape or correct the wavefront.Environmental influences, thermal effects, biological processes, sampleholder materials and properties, the sample itself, and other sources ofaberration often degrade the performance of an optical system orinstrument. Adding an adaptive optics element to an optical system orinstrument can often correct the aberrations to achieve improvedperformance.

In astronomical imaging, it is generally understood that it is desirableto locate the adaptive optics element optically conjugate to the sourceof the aberration, which is generally a turbulent layer of theatmosphere. Using multiple adaptive optics elements in a multi-conjugateconfiguration (different adaptive optics elements are conjugated todifferent turbulent atmospheric layers), it is possible to improve theaberration correction and achieve a larger field of view than isinstantaneously correctible by a particular set of adaptive opticscorrections, a concept related to improving the size of the isoplanaticpatch. Although the definition of the isoplanatic patch differs in theliterature, the isoplanatic patch describes the similarity of wavefrontwith change in field position and is most commonly described withrespect to correcting aberration with an ‘ideal’ wavefront corrector orcorrectors. A large isoplanatic patch implies that the wavefront changesslowly with field position such that a single ideal adaptive opticscorrection, or single set of corrections on different adaptive opticselements, would be able to correct the wavefront over a large field ofview. A small isoplanatic patch implies that the wavefront changesquickly with field position such that a single adaptive opticscorrection, or single set of corrections on different adaptive opticselements, would only be able to correct the wavefront over a small fieldof view. The isoplanatic patch does not, however, indicate how well anadaptive optics system will perform in practice as the adaptive opticselement may or may not have the ability to generate a wavefrontcorrection with sufficient spatial frequency, stroke, or temporaldynamic performance.

In high numerical aperture laser scanning through a well corrected (lowaberration) objective, most prior literature has taught locating theadaptive optics element in a plane that is conjugate to the pupil planeof the objective. In ophthalmic imaging, the adaptive optics element ismost commonly located in a plane conjugate to the pupil of the eye. FIG.5 shows example pupil relay configurations. An afocal 4f relay 505, orsometimes called 4f telescope, is the most common method to achieveconjugation between a pupil plane and an adaptive optics element. The 4frelay may uses lenses 510, off-axis parabolic mirrors 515, sphericalmirrors 520, combinations of lenses and mirrors, or other optics. With a4f relay, wavefront perturbations to the beam are often relayed from theadaptive optics element 525 into the pupil plane 530 of the objective530. With a 4f relay, the relative light intensity distribution ispreserved between the two planes as desired. For the purposes of thispatent application discussion, a 4f relay can preserve the beamdiameter, expand the beam diameter, or reduce the beam diameter,depending on the focal lengths of the constituent components. Adaptiveoptics scanning systems often require beam steering in addition toadaptive optics correction. An additional pupil relay consisting of a 4ftelescope is often included in an adaptive optics scanning system torelay the pupil from an adaptive optics element 525 to a steering mirror540. If the steering mirror has two degrees of freedom, as is the casewith a fast steering mirror (FSM), both axes of rotation can becoincident with the pupil plane using the one additional 4f relay.However, FSM mirrors are often not as fast as single axis galvanometerdriven mirrors. Consequently, most AO beam scanning imaging systems usetwo separate galvo driven mirrors. It is well known that placing twoseparate galvo driven mirrors in close proximity to achieve x and yscanning results in the axis of rotation being separated by a distance.This distance may be small, but it means that it is impossible to locateboth axes precisely in the same conjugate pupil plane. In adaptiveoptics scanning systems where small perturbations to the wavefront candegrade performance, it is common to include an additional pupil relay545 between the two separate galvo driven mirrors 540 and 550. Indeed,most successful adaptive optics scanning systems demonstrated to dateuse separate galvo driven mirrors separated by 4f relays. The 4f relaybetween an x direction scan galvo mirror 550 and a y direction scangalvo mirror 540 properly aligns the center of the beam with each of thescan mirrors. A 4f relay between one of the scan mirrors 540 and theadaptive optics element 525 achieves proper beam steering on theadaptive optics element. The ordering of adaptive optics element andgalvo mirrors can change, as shown in FIGS. 5(E) and 5(F). The 4f relayscan be constructed of lenses, mirrors, or a combination of lenses andmirrors. While it may be possible to design a single 4f relay with good(diffraction limited) off-axis performance, generally the 4f relay iscomposed of optical elements with mostly positive power so it isdifficult or impossible to completely balance aberrations. Cascadingmultiple 4f relays results in the aberration contributions of thepositive powered elements to compound. Consequently, it is difficult toachieve good (diffraction limited) performance through multiple seriallychained 4f relays, as is commonly implemented. The result is that mostadaptive optics systems use long working distance lenses or mirrors toreduce aberrations with the disadvantage of a large size. Additionalimprovements have been obtained by using inconvenient out-of-planeoptical configurations to reduce compounding of aberrations, as has beenshown when using off-axis spherical mirrors. The serial chaining of 4frelays has been a common method for conjugating the adaptive opticselement to the pupil plane of the objective and for properly steeringthe beam. However, the serial chaining of 4f relays suffers from a largesize and off-axis aberrations when using lenses or mirrors, andchromatic aberrations and dispersion when using lenses. Dispersionincreases with increasing thickness or number of glass elements.Dispersion is problematic when using short pulsed lasers because thepulses are dispersively broadened in time. An embodiment of the presentinvention addresses these significant shortcomings of prior art designs.An embodiment of the present invention enables a very compact andflexible adaptive optics scanning system with little to no: dispersion,chromatic aberration, and off-axis aberration for improved optical andimaging performance.

Applications of Embodiments of the Present Invention

An embodiment of the present invention is an adaptive optics scanningsystem. In a scanning optical system, light is scanned across a sample.Scanning optical systems can be used for a wide range of imaging,processing, manipulation, or characterization applications.

FIG. 1 shows examples of several imaging modalities and systems that canbe used with and embodiment of the present invention. It will beunderstood that other imaging modalities and systems not shown can alsobe used with an embodiment of the present invention. A commonapplication scans light across the sample for the purposes of learningsomething about or measuring a characteristic of the sample. Forexample, in one embodiment, the adaptive optics scanning system performsimaging of the sample. The imaging may be performed by confocal,multiphoton, second harmonic, reflected light, fluorescent, scatteredlight, or any other method of imaging a sample with a scanned beam oflight. The imaging may be one dimensional (1D), two dimensional (2D),three-dimensional (3D), or possibly 1D, 2D, or 3D as a function of timeto image dynamic processes. The imaging may be wavelength selective andpossibly multicolor or multichannel, such as is often performed influorescent imaging. A more general form of imaging seeks to obtainspectroscopic information about the sample. In one embodiment, theadaptive optics scanning system performs spectroscopy of the sample.Often, a scanning optical system is used to obtain material specificinformation about the sample, such as biological cell type, as iscommonly performed in fluorescent imaging, or scattering properties of asample, as is commonly performed with optical coherence tomography(OCT). Other applications are only concerned with the shape or profileof the sample. In one embodiment, the adaptive optics scanning systemperforms profilometry. In general, it is desirable that imaging orcharacterization of a sample be non-destructive and not change thesample itself. Often, however, photobleaching, heating, or other samplechanging phenomena occur as a byproduct of imaging. Other applicationsseek to specifically modify or affect the sample with the scanned beam,such as in laser machining, ablation, stimulation, heating, or opticalmanipulation. In one embodiment, the adaptive optics scanning systemperforms processing of the sample. In another embodiment, the adaptiveoptics scanning system performs manipulation of the sample. In anotherembodiment, the adaptive optics scanning system performs profiling ofthe sample. In another embodiment, the adaptive optics scanning systemperforms stimulation of a region of the sample. In another embodiment,the adaptive optics scanning system performs heating of a region of thesample.

FIG. 1(A) shows an optical layout for an optical coherence tomography(OCT) system. In one embodiment, the adaptive optics scanning imagingsystem performs optical coherence tomography (OCT). When performing OCT,an embodiment of the present invention may further comprise aninterferometer 110, a sample path 115, and a reference path 120 forobtaining an interferometric OCT signal from the sample 145. Scanners135 and an objective lens 140 allow a focused spot of light to bescanned across the sample 145. OCT can be performed using a variety ofmethods, include time domain, spectral/Fourier domain, or sweptsource/Fourier domain, sometimes referred to as optical frequency domainimaging (OFDI). OCT can also be performed using a high numericalaperture objective 150, called optical coherence microscopy (OCM). InOCT, low numerical aperture objectives are often used to providesufficient depth of field because information is often obtained along arelatively long depth range of an A-scan. The definition of high vs. lownumerical aperture is somewhat subjective. For the purposes of thisapplication, high numerical aperture refers to apertures commonly foundin commercial microscope objectives. FIG. 1(B) shows an optical layoutfor the sample path of an OCM system that would be connected to an OCTinterferometer. Collimated light is directed to a scanner 155 andthrough a scan lens 160 and tube lens 165 to the objective 150. In oneembodiment, the adaptive optics scanning system performs opticalcoherence microscopy (OCM). When performing OCM imaging, an embodimentof the present invention may further comprise an interferometer, asample path, and a reference path for obtaining an interferometricOCT/OCM signal and a high numerical aperture objective 150 for obtainingfine resolution sample data. One common application of OCT is imagingthe eye 170, as shown in FIG. 1(C). In one embodiment, the adaptiveoptics scanning system performs OCT of an eye 170. The retina is themost common part of the eye imagined with OCT, however imaging of theanterior eye, crystalline lens, and cornea can also be performed.

In another embodiment, the adaptive optics scanning system performsconfocal imaging. An example confocal imaging system is shown in FIG.1(D). When performing confocal imaging, the adaptive optics scanningsystem may further comprise a beam splitter or dichroic mirror 175 anddetector 180 and confocal pinhole 185 to achieve depth sectionedfluorescence or reflectance imaging. Sometimes the end of a single modeor multimode fiber is used as a confocal pinhole. A scanning laserophthalmoscopes (SLO) is a variation of confocal imaging that is usefulfor imaging the eye 190. An example SLO imaging system is shown in FIG.1(E). In one embodiment, the adaptive optics scanning system is an SLOsystem. An embodiment of the present invention can also be used withnonlinear imaging modalities. An example multiphoton/second harmonicimaging system is shown in FIG. 1(F). In one embodiment, the adaptiveoptics scanning system performs two-photon imaging. When performingtwo-photon imaging, the imaging system may further comprise a dichroicmirror 194 in the light path and the detector 735 measures ballistic andmultiply scattered fluorescent or emitted light from the sample.Three-photon and other multiphoton imaging can also similarly beperformed. In one embodiment, the adaptive optics scanning systemperforms multi-photon imaging. When performing multi-photon imaging, theadaptive optics scanning system may further comprise a dichroic mirror194 in the light path and the detector 197 measures ballistic andmultiply scattered fluorescent or emitted light from the sample. Manymultiphoton imaging systems can also be used for second harmonicimaging. In one embodiment, the adaptive optics scanning system performssecond harmonic imaging. In another embodiment, the adaptive opticsscanning system performs fluorescent imaging. More generally, anembodiment of the present invention can be used for a wide range ofapplications where a light beam is scanned on or in a sample andinformation about the sample obtained by collecting light from thesample. In addition to fluorescent and nonlinear imaging, more standardreflection and transmission imaging can be performed. In one embodiment,the adaptive optics scanning system performs reflection imaging. Inanother embodiment, the adaptive optics scanning system performstransmission imaging. Most imaging applications use a single channel ofspectral detection or a small number of spectral channels that aresufficient to differentiate sample characteristics. Other applicationsseek to spectrally resolve regions of the sample using spectroscopy. Inone embodiment, the adaptive optics scanning system performsspectroscopy. When performing spectroscopy, the adaptive optics scanningsystem may further comprise a spectrometer for resolving a spectralcontent of the light from the sample.

There are many laser scanning applications that can benefit fromadaptive optics to achieve improved performance. Therefore an embodimentof the present invention may be used on a wide range of samplesassociated with biological, medical, industrial, and research fields.Some example samples include: a biological specimen, animal, portion ofan animal, human, portion of a human, plant, portion of a plant, tissue,living tissue, preserved tissue, stained tissue, a biological organ, abiopsy specimen, an eye, a portion of an eye, a brain, a portion of abrain, or skin. Other example samples comprise: a mechanical component,an electrical component, an optical component, a fabricated component,an assembly of components, a material specimen, a semiconductorcomponent, a semiconductor material specimen, a metal component, a glasscomponent, a plastic component, an inanimate organic specimen, a crystalspecimen, or a mineral specimen. More generally, samples that can beused with an embodiment of the present invention would be characterizedby a property of the sample. The sample can be characterized withrespect to dimensional properties. The sample can be characterized withrespect to mechanical properties. The sample can be characterized withrespect to optical properties. The sample can be characterized withrespect to fluorescent properties. The sample can be characterized withrespect to reflection properties. The sample can be characterized withrespect to transmission properties. The sample can be characterized withrespect to index of refraction. The sample can be characterized withrespect to scattering properties. The sample can be characterized withrespect to dispersive properties. The sample can be characterized withrespect to spectroscopic properties. The sample can be characterizedwith respect to polarization properties. The sample can be characterizedwith respect to thermal properties.

The source of the aberrations in an embodiment of the present inventioncan come from sources internal to the adaptive optics scanning system orexternal to the adaptive optics scanning system, as shown in FIG. 2. Inone embodiment, the aberrations come from packaging 205 around acomponent 210 that is the sample, as shown in FIG. 2(A). The aberrationsmay originate from a glass window or coverslip 215 above the sample 220,as shown in FIG. 2(B). The aberrations may come from the sample orspecimen itself 225, as shown in FIG. 2(C). The aberrations may comefrom a portion of the eye 230, including the cornea 235 or crystallinelens 240, as shown in FIG. 2(D). Focusing converging light through asurface with index of refraction mismatch, such as an interface betweenan emersion fluid, glass coverslip, or the sample itself, introducesspherical aberration. Inhomogeneity of the sample may introduce otheraberrations. Thus, aberrations may change with depth, as illustrated inFIGS. 2(E-F) or with lateral position, as illustrated in FIG. 2(G).Aberrations cause distortion to the wavefront. One embodiment of thepresent invention uses the adaptive optics element(s) to compensate foraberrations in the sample. One embodiment of the present invention usesthe adaptive optics element(s) to compensate for aberrations from asample holder, which could be packaging, a coverslip, a window, a tube,a container, or any other material, object, fluid, or surface in contactwith or in between the sample and the imaging system. The imaging systemitself may have residual system aberration. One embodiment of thepresent invention uses the adaptive optics element to compensate forresidual aberrations within the imaging system.

General Description

An embodiment of the present invention is an adaptive optics scanningsystem. A schematic diagram of an embodiment of the present invention isshown in FIG. 7. One embodiment of the present invention comprises anemission source 705 for generating light, the light being directedthrough the adaptive optics scanning system to a sample 710, one or moreadaptive optics element(s) 715, the adaptive optics element(s) 715affecting the wavefront, affecting the intensity, or affecting both thewavefront and intensity of the light, a beam projection module 720, thebeam projection module 720 operating with four or more axes of motionand controlling an angle and position of the light to preferentiallyinterface the adaptive optics element 715 by creating or accommodating abeam pivot point at or near the adaptive optics element(s) whilescanning the light across the sample 710, a controller 725 forcontrolling motion trajectories of the axes in the beam projectionmodule 720, sample delivery optics 730, the sample delivery optics 730appropriately conditioning and directing the light to the sample 710,one or more detector(s) 735, the detector(s) 735 measuring light fromthe sample 710.

FIG. 7(A) shows an example embodiment in which the detector 735 islocated after, or is separate from the sample delivery optics 730. Oneexample of an embodiment in which the detector 735 is located after thesample delivery optics 730 would be a multiphoton imaging system inwhich the detector 735 receives light from the sample 710 directly, asis sometimes used when imaging thin samples or when detectors arearranged around the sample, but do not share an optical path with thesample delivery optics 730. The positioning of the detector 735 in FIG.7(A) after the sample 710 only indicates the path of the light and doesnot indicate where the detector 735 is spatially located relative to thesample 710 and sample delivery optics 730 in practice. Other embodimentsand imaging modalities can also use a configuration where the detector735 does not share an optical path with the sample delivery optics 730.FIG. 7(B) shows an example embodiment in which the detector 735 receiveslight from at least a portion of the sample delivery optics 730. Anexample embodiment in which the detector 735 receives light from atleast a portion of the sample delivery optics 730 is multiphoton imagingin which the light is collected through the microscope objective,patient interface optics, scan lens, or other sample delivery optics730. FIG. 7(C) shows an example embodiment in which the detector 735receives light from the beam projection module 720, possibly withadditional components between the beam projection module 720 anddetector 735. Example embodiments in which the detector 735 receiveslight from the beam projection module could be certain configurations ofOCT, confocal imaging, profiling, or spectroscopy. Other positions ofthe detector 735 that are not shown are possible. The detector 735 canbe located to receive or pick off light anywhere along the optical path,or can be located separate from the optical light delivery system.

As shown in FIG. 7, an embodiment of the present invention includes anemission source 705. The type of emission source used in the adaptiveoptics scanning system is selected to be compatible with the scanningapplication. Depending on the imaging modality, the emission source 705can generate light with a diode, a laser, a pulsed laser, a tunablelaser, a wavelength swept laser, a femtosecond laser, a fiber laser, avertical-cavity surface-emitting laser (VCSEL), a wavelength tunableVCSEL, a plasma light source, a halogen lamp, a mercury lamp, anincandescent lamp, or a supercontinuum source. Other emission sources705 are possible and included in an embodiment of the present invention.

The requirements on the light delivery from the emission source 705depend on the application. Possible emission source characteristics areshown in FIG. 8. For example, a multi-photon imaging system maypreferentially use a collimated beam from the emission source, while aconfocal imaging or OCT system may preferentially use light deliveredfrom a single mode or multi mode fiber. The present invention includesembodiments where the emission source includes optics for collimatinglight from a point source or small area emitter. In many cases, lightfrom the emission source is collimated. Collimated or predominatelycollimated light is emitted from a titanium sapphire laser, among otherlight sources. An emission source 805 emitting collimated light is shownin FIG. 8(A). In one embodiment of the present invention, light from theemission source 805 is collimated. Light emitted from a point sourcethat passes through a lens exiting the emission source may form aconverging beam. In another embodiment of the present invention, thelight from the emission source 810 is converging, as shown in FIG. 8(B).Light from a point source or small area emitter may form a divergingbeam, as shown in FIG. 8(C). In one embodiment of the present invention,the light from the emission source 815 is diverging. For manyapplications, such as OCT and confocal imaging, it is desirable that thelight be delivered with a fiber optic cable, as shown in FIG. 8(D). Inone embodiment of the present invention the light from the emissionsource 820 is fiber coupled. Further, it is sometimes desired that thefiber optic cable 825 be single mode, as is the case for OCT and someimplementations of confocal imaging. In one embodiment of the presentinvention the light from the emission source is fiber coupled into asingle mode fiber. Light from the emission source can have very manyshapes and light intensity distributions, all of which are included inan embodiment of the present invention. It is common that light from alaser or point source has a beam cross section 830 that is predominatelycircular, as shown in FIG. 8(E). In one embodiment of the presentinvention includes the light from the emission source is a beam with across section 830 that is predominately circular. Light from a lasersource and other sources is often generally Gaussian in lightdistribution, as shown in FIG. 8(F). In one embodiment of the presentinvention the light from the emission source is a beam that ispredominately Gaussian in intensity distribution. Different applicationsrequire different performance specifications for the emission source. Anembodiment of the present invention includes implementations where theemission source 705 generates light with broadband spectral content andemits over a range of wavelengths (greater than approximately 2 nm).Applications that often use a broadband light source include OCT,multiphoton microscopy, confocal microscopy, fluorescent microscopy(using arc lamps, incandescent lamps, or LEDs), certain spectroscopyimplementations, and others. Broadband light sources include swept lightsources or light sources that emit continuous or pulsed broadbandemission. An embodiment of the present invention includesimplementations where the emission source 705 generates light withnarrowband spectral content and emits over a narrow range of wavelengths(less than approximately 2 nm). Applications that often use a narrowband light source are confocal and fluorescent imaging (using laserlight sources), certain types of profilometry, certain types ofspectroscopy, and others.

An embodiment of the present invention includes an adaptive opticselement, also equivalently referred to as a wavefront corrector. Thereare many possible adaptive optics elements that can be used in anembodiment of the present invention, a subset of which are shown in FIG.3. An embodiment of the present invention may use an adaptive opticselement that is a deformable mirror 305, 310, 315, 320, 325, 330, and335, a liquid crystal spatial light modulator 340 and 345, a liquidcrystal device 340 and 345, a deformable mirror with continuousfacesheet 305, 315, 320, 325, 330, 335, a segmented deformable mirror310, a spatial light modulator 340 and 345, or other active and multiactuator or channel optical element that can affect the wavefront,affect the intensity, or affect both the wavefront and intensity of thelight. The arrangement of the actuators in the adaptive optics elementcan vary depending on the design of the adaptive optics element. Commonactuator layouts are grid patterns, honey-comb patterns, concentriccircles, radially aligned actuators, circle or arc segment actuatorlayouts, and others. In one embodiment of the present invention, theadaptive optics element(s) 715 is a deformable mirror. In one embodimentof the present invention, the adaptive optics element(s) 715 is a liquidcrystal spatial light modulator.

Many adaptive optics systems use pupil relays as shown in FIG. 5 toproperly manage the requirements of beam pivot locations relative toscanner locations in the optical system. A traditional pupil relayperforms a true relay of the pupil field and is used as the opticallinkage between steering mirrors, the adaptive optics element, and thesample delivery optics or the sample itself in most existing adaptiveoptics systems. The preferred embodiment of the present invention uses adifferent approach. Because the beam diameters in laser based imagingsystems are on the order of 0.5 mm to several cm and the beam quality isgenerally quite good, the divergence of the beam and associated changesin wavefront and intensity distribution over the path lengths of theinstrument are negligible. Indeed, many existing two-photon microscopesare built using a commercially available laser source (e.g., CoherentChameleon®) mated to a commercially available two photon microscope(e.g. Prairie Technologies Ultimal®) in such a way that the collimatedbeam emerging from the laser source and entering the microscopetraverses a distance that differs from installation to installationbased on what is convenient to the layout and space available to the labor site of installation. The beam maintains quality and is predominatelyunchanged over the cm to meters of collimated propagation so thatcareful control of distances and planes is not necessary. In otherwords, the high quality laser beam propagates with an essentiallystationary (unchanging) intensity and wavefront over microscopeinstallation scale (cm to several meters) distances. With this in mind,it is helpful to revisit the standard AO configuration in which a pupilrelay (afocal 4f telescope) is used to image the pupil plane of onesteering mirror 605 to the pupil plane of another steering mirror 610,as shown in FIG. 6. A first effect of the pupil relay (afocal 4ftelescope) 615 is to create a virtual pivot point at the mirror plane620 so that the beam is always directed towards the center of the mirrorfor all incoming and outgoing beam angles. A second effect of the pupilrelay is to sample and relay the optical field from one pupil plane 605at a first steering mirror to another pupil plane 610 at a secondsteering mirror. In this way, the optical wavefront and intensitydistribution are relayed from the first pupil plane 605 to the secondpupil plane 610, even if the wavefront is not flat and the intensitydistribution complex. For a scanning adaptive optics system, the firsteffect of creating the virtual pivot point is necessary, while thesecond effect of relaying the wavefront and intensity distribution isnot necessarily required. If the beam is sufficiently large in diameter,predominately collimated, predominately of planar wavefront, andpredominately of Gaussian intensity distribution, the beam willpropagate between flat mirror reflections essentially unchanged. It istherefore not necessary to formally relay the pupil planes, but ratherit is desirable to simply create virtual pivot points of the beam. Thesame reasoning holds when relaying from the second pupil plane 610 tothe plane of the adaptive optics element 625, as is commonly performedwith a second pupil relay 630. Consider a beam with an intensitydistribution and wavefront that nominally remains predominately constantover cm to meters of propagation distance, such as the beams described.FIG. 9 shows that it is possible to use a method of creating a virtualpivot point with a stationary position, but a variable beam angle usinga pair of rotating mirrors instead of lenses or concave mirrors as wouldbe used in a traditional 4f design. In the neutral (zero) position asshown in FIG. 9(B), the beam initially propagates from left to right,reflecting off a first steering mirror 905. The beam then propagatesupwards and to the left, reflecting off a second steering mirror 910.The second steering mirror directs the beam to propagate towards theright, where it intersects a plane 915 at a particular location. Byappropriately adjusting the steering mirror angles as shown in FIG.9(A), the same optical configuration can create a downwards beam anglethat intersects the plane 915 at the same location as in the neutralposition shown in FIG. 9(B). Similarly, a different set of steeringmirror angles, as shown in FIG. 9(C), generates an upwards beam anglethat intersects the plane 915 at the same location. FIG. 9(D)superimposes the three configurations shown in FIGS. 9(A-C) and showsthat proper adjustment of the steering mirror angles allows a plane 915of predominately stationary beam intensity to be formed with adjustablebeam angle around a pivot point. Setting proper angles on the mirrorsallows intermediate beam angles to be generated that all rotate aroundthe same pivot point. This optical construct can be used to satisfy therequirement of creating a virtual pivot point to be used as the linkagebetween the beam steering mechanisms and the adaptive optics element inan adaptive optics scanning system in place of the more traditionalpupil relay (afocal 4f telescope), but has significant advantages of nooff-axis aberration, no dispersion, and a compact size. FIG. 9(D) showsa beam entering from the left and a beam of stationary intensityposition but variable angle being formed to the right of the beamsteering mechanism in a plane 915. FIG. 9(E) shows that the same opticalconstruct can also be used to accept light from a plane 920 ofstationary beam intensity position, but variable beam angle and generatea second plane 925 of stationary beam intensity position, but variablebeam angle to the right of the beam steering mechanism by using a firststeering mirror 935 and second steering mirror 940 oriented atappropriate angles. Further, the flexibility of the arrangement allowsarbitrary and programmable placement of the position and the angle ofthe output beam. For example, the plane 945 in FIG. 8(D) shows achanging beam position with changing beam angle. The beam position andbeam angle are fully programmable within the working apertures of themirrors and can be pre-programmed or recalculated on the fly to createarbitrary beam position and beam angle trajectories during the scan,which may or may not pivot around the same point. The two basicfunctionalities illustrated in FIGS. 9(D-E) can be used as buildingblocks for a compact scanning mechanism in an adaptive optics scanningsystem. Most beam scanning systems scan in two directions, X and Y,across the sample. FIG. 10 shows that it is possible to connect an Xbeam scanning mechanism 1005 consisting of two rotating mirrors with a Ybeam scanning mechanism 1010, also consisting of two rotating mirrors.The two beam scanning mechanisms are oriented in orthogonal directions(rotated 90 degrees) and project to the same stationary point in a plane1015. In this example, the second pair of rotating mirrors 1020 and 1025fits between the stationary point in the plane 1015 and the first pairof rotating mirrors 1030 and 1035 in such a way that the output of thefirst rotating mirror pair becomes the input to the second rotatingmirror pair. FIG. 11 shows an example configuration based on thisprinciple that illustrates x and y scanning using four galvanometers(galvos) to actuate the steering mirrors. Other relative ordering andarrangement of the mirrors are possible. The collection of mirrors andactuators that generate the programmable position and angle of the beamis referred to as a beam projection module 720 in this patentapplication.

An embodiment of the present invention of an adaptive optics scanningsystem includes a beam projection module for generating a pivot locationfor the beam at an appropriate location in the optical system. The beamprojection module has four or more axes of motion that affect mirrors toproperly guide the beam. One embodiment of the present invention uses anarrangement where at least one axis of the beam projection module isrotational. As shown in FIG. 11, one embodiment of the present inventionincludes a beam projection module that comprises four galvanometerdriven mirrors. The ordering of the galvos can be optimized to thespecific imaging application. One embodiment of the present inventionuses an optical layout in which the two x-axis galvos precede the y-axisgalvos. In another embodiment, the two y-axis galvos precede the twox-axis galvos. In another embodiment the axis are split such that afirst x and y galvo precede a second x and y galvo. Other mirror andactuator configurations are also possible. For example, FIG. 12(A) showsa beam projection module comprised of two fast steering mirrors (FSMs)1205 and 1210 to project one external pivot point 1215. FIG. 12(B) showsa beam projection module comprised of two fast steering mirrors (FSMs)1220 and 1225 that accepts light from an external beam pivot point 1230and projects an external beam pivot point 1235. Combinations of an FSMand galvos are also possible. One embodiment of the present inventionincludes a beam projection module comprising at least one fast steeringmirror (FSM). Another more specific embodiment of the present inventioncomprises a beam projection module that uses two fast steering mirrors,each fast steering mirror having two axes of rotation. Other two-axes,single mirror beam steering elements can also be used as described withthe FSM, such as MEMS mirrors, gimbaled mirrors, piezo driven tip-tiltmirrors or other tip-tilt mirror mechanisms. In another embodiment, thebeam projection module comprises at least one galvanometer drivenmirror. Not all of the actuators and mirror motions have to berotational. It is possible to combine rotational and translationalactuators and mirror movements to accomplish the goal or projecting abeam to a programmable position and beam angle. FIG. 12(C) shows how afirst rotational mirror 1240 can be combined with a second translationalmirror 1245 to create a beam projection module that generates anexternal beam pivot 1250. The ordering of rotational to translationalaxis can differ. FIG. 12(D) shows how a first translational mirror 1255can be combined with a second rotational mirror 1260 to create a beamprojection module that generates an external beam pivot 1265. Moregenerally, one embodiment of the present invention operates with atleast one axis of the beam projection module being translational.Another embodiment of the present invention comprises a beam projectionmodule that uses a combination of rotational and translational axes ordegrees of freedom. Other beam steering devises are possible. Oneembodiment of the present invention operates with the beam projectionmodule comprising at least one of the following list: steering mirror,acousto-optic deflector, rotating polygon, electro-optic beam deflector,electro-optic prism, thermo-optic prism, diffractive array, mechanicallyscanned mirror, mechanically scanned mirror driven by a motor,mechanically scanned mirror driven by a stepper motor, a mechanicallyscanned mirror driven by a galvanometer, a MEMS mirror, anacoustic-optic modulator, or a liquid crystal device.

The angles or positions of the mirror must be controlled to generate thedesired beam projection output. Many actuators have associated feedbackcontrol systems such that a position command is used as an input tocommand the actuator and the control system acts to track the commandedposition. For example, a galvo system may use a capacitive or opticalencoder to measure the position of the galvo angle. The measuredposition is compared to a commanded position to generate a positionerror. The position error is processed by a feedback controller, forexample a proportional-derivative-integral (PID) controller or fullstate feedback controller, to generate a corrective action to be appliedto the actuator in the galvo. In this way, commands to the mirror areexecuted up to bandwidth, acceleration, and velocity limits of theactuator and controller. These localized feedback control systems managethe low level position control of the actuators. Other actuators respondwell to open loop position commands, such as MEMS devices and piezoactuators. One embodiment of the present invention uses closed loopcontrol for at least one axis in the beam projection model. Anotherembodiment of the present invention uses open loop control for at leastone axis in the beam projection module. However, regardless of thelocalized actuator control scheme, the positions between the differentactuators and mirrors in an embodiment of the present invention must becarefully coordinated to generate the desired beam steering effect.

Coordination between the different axes in the beam steering module isperformed by a controller 725 for controlling the motion trajectories ofthe axes in the beam projection module. The controller generatesposition commands to the individual actuators to coordinate the motion.In the preferred embodiment, the coordination is performed by aprocessor or circuit that can execute code, logic, or instructions togenerate the desired position commands. The processor can be amicroprocessor, a microcontroller, a digital signal processor (DSP),field programmable gate array (FPGA), application specific integratedcircuit (ASIC), or any other processor that can perform digitalcalculations. A digital processor is preferable because nonlinearcalculations can be performed, there is adjustability and flexibility incalculation, and there is often spare processor capability alreadyavailable in many imaging systems. However, analog circuits can also beused to perform the control. Depending on if the actuators for themirrors in the beam projection module are controlled in an open loop ora closed loop (feedback) manner, the controller may also comprise theclose loop controllers in addition to the processor that generates themotion trajectories for each of the axis in the beam projection module.In either case of open or closed loop actuator control methods, thecontroller generates motion trajectories and is for controlling themotion trajectories of the axes in the beam projection module. In thepreferred embodiment of the present invention coordination between thedegrees of freedom in the beam projection module is controlled throughelectronic signals to the actuators or active elements. In oneembodiment, the actuators for any given scan axis are coupled such thatthe desired output command for each individual axis is determined by asingle input parameter. In another embodiment of the present invention,coordination between the degrees of freedom in the beam projectionmodule is controlled through a mechanical linkage. An imaging systemgenerally scans a spot on the specimen and many scan trajectories arepossible. One embodiment of the present invention uses the beamprojection module 720 to scan the mirrors with trajectories that causethe light beam to trace a raster scan pattern 1705 on the sample 710.

An embodiment of the present invention includes optics for deliveringthe light to the sample 710, called sample delivery optics 730. Mostsamples require that an objective lens focus the light to or into thesample. One embodiment of the present invention includes sample deliveryoptics comprising a microscope objective. More generally, one embodimentof the present invention uses sample delivery optics that direct thelight towards the sample in a converging beam with a numerical aperture(NA) to achieve a desired resolution in the sample. Other samplesinclude their own optics or optical surfaces, such as the biological eyeor a camera system, which have different requirements on thecharacteristics of the light delivery such that a collimated or nearlycollimated beam is preferable for delivery of light to the sample. Oneembodiment of the present invention uses sample delivery optics thatdirect the light towards the sample in a predominately collimated beamwith a pivot point located at or near a pupil plane within the samplesuch that optical properties of the sample focus the light at a desiredimaging plane. More specifically, one embodiment of the presentinvention uses a predominately collimated beam directed into an eye, thepivot point of the beam being located at or near the pupil of the eyesuch that the light is focus at or near the retina 245 in the eye. Thesample delivery optics are used for conditioning and directing the lightto the sample, where conditioning refers to generating the appropriatecollimation, convergence, or divergence of the beam, generating theappropriate beam diameter, generating the appropriate numericalaperture, generating an appropriate intensity profile, generating anappropriate spot size, generating an appropriate spot shape, generatingan appropriate wavefront, or any other way of affecting a light beam topreferentially interact with the sample.

An embodiment of the present invention includes a detector 735 fordetecting light from the sample 710. In one embodiment of the presentinvention, the detector 735 is a line scan camera for performingspectral/Fourier domain OCT. In another embodiment of the presentinvention, the detector 735 comprises a high speed photodiode toimplement unbalanced detection or two high speed photodiodes toimplement balanced detection for performing swept source/Fourier domainOCT. In another embodiment of the present invention, the detector 735comprises a photomultiplier tube (PMT) or avalanche photo diode. Morespecifically, one embodiment of the present invention uses a detectorthat comprises a photomultiplier tube (PMT) or avalanche photo diode forperforming two-photon, multi-photon, or second harmonic imaging. Anotherembodiment of the present invention uses a detector 735 comprising aphotomultiplier tube (PMT), photo diode, or avalanche photo diode forperforming confocal imaging. In yet another embodiment of the presentinvention, the detector is a spectrometer for resolving a spectralcontent of light from the sample. Another embodiment of the presentinvention uses a detector that records information in the light from thesample with a photo-chemical reaction, as is used in film. Anotherembodiment of the present invention uses a detector that recordsinformation in the light from the sample with a thermal sensor. Inoptical tweezer systems, the object under manipulation is oftenmonitored with a camera, for example a charge-coupled device (CCD) orComplementary metal-oxide-semiconductor (CMOS) array. Applying anoptical force to the object under manipulation and monitoring theresponse of the object with a camera can indicate the strength of theoptical trapping force. Measurement of the optical trapping forcedepends on aberrations in the system and an adaptive optics element canbe optimized to maximize trapping force. In one embodiment of thepresent invention, the detector 735 is a camera. In another embodimentof the present invention, the detector 735 is a wavefront sensor. Inanother embodiment of the present invention, the detector 735 measuresan intensity of light from the sample. The detector 735 can be locatedin different locations along the optical path, consistent with theapplication, or can be located separate from the optical systemdelivering light to the sample.

Multiphoton Microscopy Embodiment

An embodiment of the present invention can be used for adaptive opticsmulti-photon imaging. FIGS. 13-21 teach an embodiment of the presentinvention that performs two-photon microscopy and FIGS. 22 and 23 showexperimental adaptive optics two-photon results. The emission source 705in the prototype is a commercially available Titanium-Sapphirefemtosecond laser (Thorlabs Octavius-2P), as shown in FIG. 21(C). Theadaptive optics element 715 in the prototype is a commercially availableMEMS deformable mirror with 140 actuators, gold coating, grid actuatorlayout, electrostatic actuation, and 4.4 mm active area (BostonMicromachines Multi-DM) 2105, as shown in FIG. 21(B). The beamprojection module 720, diagramed in FIG. 13, comprises four commerciallyavailable galvanometers (Cambridge Technology 6210H X-Y scanners). Eachpair of X-Y scanners is controlled by an analog controller sold with thegalvos that performs close loop control of the galvo angle position. Ananalog voltage signal is used as input to the controller to command adesired galvo position angle. As shown in FIG. 13(A), light enters thebeam projection module 1305 and reflects off a first steering mirror1310, labeled X mirror 1. Light then travels to a second steering mirror1315, labeled X mirror 2. X mirror 1 and X mirror 2 work together tocontrol the angle and position of the beam in the X scanning direction.Light from X mirror 2 travels to a steering mirror 1320, labeled Ymirror 1, which reflects and directs the light to a steering mirror1325, labeled Y mirror 2. Y mirror 1 and Y mirror 2 work together tocontrol the angle and position of the beam in the Y direction. Note thatthe X and Y directions are chosen for convenience of illustration andthat the ordering of X and Y is interchangeable. The commercial X-Yscanner kit contains a pair of galvos with a small and a large mirror,thus two kits contain two galvos with small mirrors and two galvos withlarge mirrors. The dynamic performance of the galvo with small mirror isdifferent than the performance of the galvo with a large mirror. It istherefore desirable in the context of the beam projection module tomatch mirrors size within each scan axis. In the specific embodimentshown, it is desirable to use the two small mirrors 1310 and 1315 in thefirst stage of the beam projection module (labeled X mirror 1 and Xmirror 2 in FIG. 13) and the two large mirrors 1320 and 1325 in thesecond stage of the beam projection module (labeled Y mirror 1 and Ymirror 2 in FIG. 13). Matching mirror sizes within an stage means thatscanning within the stage is simplified because the dynamic performanceof the two galvos are similar so they will respond similarly to inputcommand voltage trajectories. Further, placing the two small mirrorsbefore the two large mirrors is advantageous because the beam from Xmirror 1 and X mirror 2 has been directed off-axis, requiring a larger Ymirror 1 and Y mirror 2 surface to receive the off-axis beam, as shownin FIG. 14(B). Light reflected from Y mirror 2 exits the beam projectionmodule and travels to the deformable mirror 1330, which has a highlyreflective surface to reflect the light towards the output of the beamsteering module. Changing the angles of the steering mirrors allows thebeam angle incident on and reflected from the deformable mirror 1330 tobe changed while at the same time maintaining centration of the beam onthe deformable mirror 1330. The output rays depicted in FIG. 13(B) andlabeled Direction 1, Direction 2, and Direction 3 illustrate thisprinciple of beam steering that enables a compact interface to thedeformable mirror 1330 and scanning of the beam on the sample. FIGS.13(A) and 13(B) show a projection view of FIG. 13(B) and FIG. 13(D) anisometric view of the beam steering module and adaptive optics elementassembly. FIGS. 14(A and B) show a solid model drawing of the beamsteering module 1405 in which the steering mirrors and the deformablemirror surface are shown relative to the input and output beams. FIG. 15shows the steering mirror locations and angles of the prototypeembodiment.

The controller 725 for controlling the motion trajectories of the axesin the beam projection module 720 of this embodiment comprises softwarecode running on a PC computer 1605 (Dell desktop PC), a digital toanalog converter (DAC) board 1610 (National Instruments PCIe-6323), andthe analog controllers for the galvos 1615 and 1620, as shown in FIG.16(A). The PC computer 1605 contains a central processing unit 1625(CPU). An algorithm 1630 is executed by the CPU 1625 to generate mirrorangle trajectories for each of the galvos to accomplish scanning adesired optical spot trajectory in the imaging field (imaging plane).The mirror angle trajectories are stored as an array in computer memory.To perform the scan, the mirror angle trajectories represented asdigital data are output by the DAC board at a fixed rate of 100,000samples per second at 16 bits of DAC resolution as an analog outputvoltage on each of four channels, Ch1 through Ch4. The outputs of Ch1through Ch4 are connected by electrical cabling 1635 to the inputs ofthe two galvo controllers, 1615 and 1620. Each of the two galvocontrollers 1615 and 1620 performs close loop control for two channelsof the four galvos 1640, 1645, 1650, and 1655 in the beam projectionmodule 1660. FIG. 16(B) shows how a scan trajectory is generatedreferenced to the imaging field (imaging plane) coordinate system by ascan trajectory generator 1665 as a first step. Based on the geometry ofthe beam projection module, a set of galvo angles that are required togenerate the desired scan pattern in the field can be calculated by amirror angle calculation 1670. The mirror angle calculation takes asinput the X and Y field positions and generates corresponding X1 galvo,X2 galvo, Y1 galvo, and Y2 galvo steering mirror command angles. The X1galvo, X2 galvo, Y1 galvo, and Y2 galvo commands that are digitallyrepresented and stored in computer memory are transmitted to the DAC1675 to be converted to analog output signals to be output from Ch1,Ch2, Ch3, and Ch4.

FIG. 17 shows details of the scan patterns used in the imaging field ofthe experimental apparatus. A raster scan pattern 1705 consists of arepeated sequence of left to right imaging paths that scan the opticalspot across the sample at constant velocity in the x direction, as shownin FIG. 17(A) and FIG. 17(B). As the optical spot is scanned across thesample, an A/D converter reading light intensity collected from the PMTmeasures the signal from the sample, the data being used to generate arow of data in the final two photon image. At the end of each constantvelocity x direction scan of the imaging path 1710, the scan patterndefines a rapid flyback motion or path 1715 to return the spot to thestart of a new constant velocity x direction scan 1710. At the same timeas the flyback, the scan pattern defines a small upwards movement of theoptical spot in the y direction, a row stepping movement 1720, to scanthe next adjacent row. In practice, galvanometers can only track withinlimited closed loop dynamic bandwidth and are subject to oscillation andringing effects when commanded by trajectories that are not suitablysmooth and within achievable motion limits. FIG. 17B shows details ofthe forwards scanning and flyback trajectory used for raster scanning inthe prototype embodiment. The trajectory is based on half sine waveprofiles in acceleration, which is well studied and a common trajectoryused in the field of motion control and robotics to reduce undesirableexcitation of vibratory and resonant modes when there are accelerationand velocity constraints on a dynamic system. The derivative withrespect to time of the half sine acceleration is the jerk profile, whichis bounded in value. The integral with respect to time of the half sineacceleration profile is the velocity profile. The integral with respectto time of the velocity profile is the position profile, which is usedas the motion path in the field reference frame. FIG. 17(C) shows thescan trajectories of the imaging path and flyback for the x directionscanning in the top plot and the associated row-stepping movement in thebottom plot. The row stepping movements 1720 are also based on half-sineacceleration profiles. Other scan trajectories are possible anddesirable for imaging and optimization. FIG. 17(D) shows concentricconstant velocity imaging circles scanned with respect to field positionwith each the circles being joined with small non-imaging path segments.The associated x and y field positions and field velocities are plottedin FIG. 17(E). FIG. 17(F) shows a radial cross pattern imaging scansjoined by non-imaging turnaround segments that are each optimal withrespect to galvo acceleration and velocity constraints. The associated xand y field positions and field velocities are shown in FIG. 17(G).Scanning trajectories defined in the field reference plane aretransformed into galvo coordinate system trajectories for execution.

FIG. 18(A), top, shows the galvo angles that are required to generatethe desired output angle for the x direction as determined by solvingthe ray tracing equations by numerical methods. The scan geometry wasdefined with the ZEMAX ray tracing software and the nonlinear solver(optimizer) used to calculate the steering mirror angles that generatethe desired output beam position and angle. FIG. 18(B), bottom, showsthe required galvo angles that are required to generate the desiredoutput angle for the y direction as determined by solving the raytracing equations by numerical methods. These plots representcalibration curves such that required galvo angles can be determinedfrom an input of a desired scan angle in the imaging field coordinatesystem. The curves look predominately linear, as can be seen in FIG.18(B), top, where a first order polynomial is fit to the calibrationcurve data using linear regression. FIG. 18(B), bottom, shows theresidual fit error and the nonlinearity in the calibration data. Thehigh degree of linearity in the calibration curve indicates that it ispossible to run an embodiment of the present invention with a linearcalibration curve, although there will be a small error. Higher orderfits to accommodate the non-linearity can be used for improvedcalibration performance. FIG. 18(C) shows the residual error forpolynomial fits of order 1, 3, and 5, with each increasing order showingimproved calibration curve performance. Other parameterizations andbasis can be used to represent the calibration curve, includinginterpolation methods or selection of other basis functions. A linearcalibration was used in the experimental prototype such that the voltageapplied to each galvo was: V_(galvo) _(_) _(x1)=C₁θ_(x)+C₂, V_(galvo)_(_) _(x2)=C₃θ_(x)+C₄, V_(galvo) _(_) _(y1)=C₅θ_(y)+C₆, V_(galvo) _(_)_(y2)=C₇θ_(y)+C₈, where V is the command voltage to the galvo asindicated in the subscript, θ_(x) is the x field position, θ_(y) is they field position, the odd indexed coefficients of C are scaling factors,and the even indexed confidents of C are DC offset values of thecalibration curve. Because the absolute rotational angle of the galvowithin the machined mount for the galvos was not controlled (i.e, thegalvo itself could rotate within the bore hole of the machined mountbefore being tightened with a set screw), the DC offset values weredetermined by finding the analog output voltage that centered the beamon all of the mirrors and generated an output beam centered on thedeformable mirror. The angle to voltage conversions for the scalingfactors were determined experimentally by directing a laser into thebeam projection module and measuring spot locations on a projectionscreen located a known distance from the output of the beam projectionmodule as the voltages to the galvos were changed.

The beam projection module 1905 and deformable mirror 1910 are combinedwith a scan lens 1915, tube lens 1920, and objective 1925 to form thesample delivery optics 730 of a two-photon microscope, as shown in FIG.19(A). A zoom in on the beam projection module shows a schematic of thebeam projection module described in detail in FIGS. 13-15. The steeringmirrors are angled to create an off-axis scan position with rays tracedwith ZEMAX in FIG. 15(B). FIG. 20 shows the lens prescriptions and lensspacing of the two-photon microscope. A long pass dichroic mirror (680nm-1600 nm) 2005 is placed in the excitation path to pass the longwavelengths of the laser source to the sample and reflect thefluorescent signal through a filter cube 2010 containing an emissiondichroic filter and an emission bandpass filter 2015, to the PMTdetector 2020, where the emission dichroic filter and emission bandpassfilter are chosen based on the fluorescent properties of the samplebeing imaged. The objective 2025 is a commercially available waterdipping objective (Nikon LWD 16× 0.8NA). The detector 2020 was acommercially available PMT (Hamamatusu H7422PA). Photographs of theexperimental prototype of an embodiment of the present invention areshown in FIG. 21, where the four galvos are indicated with 1-4 in FIG.21(A) and the deformable mirror (DM) 2105 indicated in FIG. 21(B). Theexperimental setup is shown in FIG. 21(C). This apparatus was used toimage a paper sample 710 on a microscope slide with an optical gelapplied to the coverslip to generate an aberration. The index ofrefraction of the gel was similar to brain tissue and the gel surfacetextured to create a phantom brain sample. FIG. 22 shows a screencapture from a software to control the prototype. An image of the sample2205 is shown. The optimized deformable mirror shape 2210 can be seen. Aplot 2215 showing the progress of the optimization is shown. Theresulting amplitudes of the basis functions applied to the deformablemirror can be seen in a plot 2220. FIG. 23 shows images of the samplewith deformable mirror flat 2305 and deformable mirror optimized 2310.The flat mirror image 2305 suffers from the aberrations from the gel.The optimized mirror image 2310 shows increased signal and improvedresolution by correcting for the aberrations generated by the gel byproperly shaping the deformable mirror.

The adaptive optics convergence algorithm was based on the algorithmpresented in “Image based adaptive optics through optimisation of lowspatial frequencies” by D. Debarre, M. Booth, and T. Wilson, Opt.Express 15, 8176-8190 (2007) and “Image-based adaptive optics fortwo-photon microscopy” by D. Débarre, E. Botcherby, T. Watanabe, S.Srinivas, M. Booth, and T. Wilson, Opt. Lett. 34, 2495-2497 (2009) whichteach and demonstrate sensorless adaptive optics algorithms andimplementation for adaptive optics two-photon optimization.

It is noted that the same optical instrument used for multiphotonimaging can also be used for second harmonic imaging with properselection of emission filters and excitation wavelength. It is alsopossible to reduce the size of an embodiment of the present invention byusing custom designed optics instead of off-the-shelf optics.

OCT Imaging Embodiment

An embodiment of the present invention can be used for adaptive opticsOCT imaging. FIG. 24 shows an adaptive optics OCT imaging system thatuses swept source OCT (SS-OCT) detection, sometimes called sweptsource/Fourier domain OCT, or optical frequency domain imaging (OFDI).The same basic interferometer 2405 design shown in FIG. 24(A) can beinterfaced to different sample delivery optics. FIG. 24(B) shows sampledelivery optics suitable for imaging an eye 2410. FIG. 24(C) showssample delivery optics suitable for imaging a sample 2415 that includesa focusing objective or scan lens 2420 that has an external pupil. FIG.24(D) shows sample delivery optics suitable for imaging with amicroscope objective 2425 or other similar objective that has a pupilinternal to the scan lens. In swept source OCT, a wavelength swept lightsource 2430 generates light with an emission that sweeps a narrowlytuned wavelength in time, as shown in FIG. 25(A). Light from theemission source 2430 is fiber coupled to a first fiber coupler 2435, asshown in FIG. 24(A). A portion of the light is split and directed to areference path or alternately called a reference arm, 2440. The otherportion of the light is split in the fiber coupler and directed to thebeam projection module (BPM), adaptive optics element (AOE), and sampleoptics 2445. Light from the sample optics is directed to a sample 2410,2415, 2450. Backscattered and reflected light from the sample 2410, 2415or 2450 is collected by the sample optics and returns through theoptical fiber. A portion of the light returning from the sample 2410,2415 or 2450 passes through the first fiber coupler 2435 to a secondfiber coupler 2455 where it interferes with light from the reference arm2440. Light from the second fiber coupler 2455 is directed to a balanceddetector 2460 which converts the light to an electrical signal for eachchannel, subtracts the signals from the channels, and generates avoltage output. The voltage output is digitized by an analog to digitalconverter (A/D) 2465 to form an interferogram 2505, as shown in FIG.25(A). The interferogram is Fourier transformed to generate thereflectivity vs. depth profile, called an axial scan or A-scan 2530.Scanning across the sample and assembling adjacent A-scans can form atwo dimensional cross sectional image, a B-scan 2535. Scanning theimaging spot over the sample in a raster scan pattern and assemblingadjacent B-scans can form a three dimensional volumetric data set 2540.It should be noted that implementations of OCT other than swept sourceOCT are also possible, including spectral domain OCT (SD-OCT), sometimescalled spectral/Fourier domain OCT, which uses a broadband light sourceand spectrometer, and time domain OCT (TD-OCT), which uses a broadbandlight source, single point detectors, and a moving mirror in thereference arm. OCT is a well developed field and there is a large bodyof literature teaching different OCT implementations including OCTsystems that use fiber optic components, OCT system that use bulk opticscomponents, OCT used for Doppler measurement, OCT used for polarizationsensitive measurement, and others. Any point scanning OCT method can beused in an embodiment of the present invention. However, in the contextof an embodiment of the present invention, swept source OCT offersadvantages over spectral domain OCT and time domain OCT because of theshort time integration and efficient sampling in the swept sourcedetection method.

One challenge that arises when using an embodiment of the presentinvention for OCT is that there is a path length change that occursduring scanning. FIG. 25(E) shows a ray trace of the beam projectionmodule for an on-axis field position and an off-axis field position.Because of the forwards and backwards reflections off the mirrors, thereis an additional optical path length introduced for the off-axis scanpositions. Further, the amount of additional optical path length changevs. field position is different for the x and y axis because of thedifferent mirror spacing, as shown in FIG. 25(F). Because theinterferogram in OCT is a function of the difference in path lengthbetween the reference arm and the sample arm, a first effect of theoptical path length change in the beam projection module is to add adistortion to the OCT image. FIG. 26(E) shows what would be expectedfrom an OCT B-scan cross sectional image 2650 of a flat mirrorreflection, while FIG. 26(F) shows distortion to the image 2655 due tothe longer path lengths at off-axis scan angles. A second effect of thechange in path length is to alter the shape of the interferogram,potentially reducing OCT instrument sensitivity, degrading axialresolution, and introduction depth measurement error. These effects canbe better understood by looking at the equations related to generatingthe interferogram. Refer to Eq. 1 below, where k_(m) is the wavenumberat sample point m, I[k_(m)] is the instantaneous photocurrent at samplepoint m, ρ[k_(m)] is the detector responsively at sample point m,S[k_(m)] is the instantaneous power on the sample at sample point m,R_(R) is the reflectivity of the reference mirror, R_(S) is thereflectivity of the sample mirror, z_(r) is the depth of the referencemirror, and z_(s) is the depth of the sample arm mirror. Equation 1 wasadapted from J. A. Izatt and M. A. Choma, Section 2.7, W. Drexler and J.G. Fujimoto Ed., “Optical Coherence Tomography: Technology andApplications”, 2008. In practice, the photocurrent, I, is generallytransformed into a voltage by a transimpedance amplifier before A/Ddigitization. A wavelength swept light source 2510 generates an emissionthat tunes the wavelength in time, as shown in the wavelength vs. timeplot in FIG. 25(A). Light travels through the OCT interferometer 2515where a photodiode converts the light intensity into current, I[k_(m)],which is transformed to a voltage output signal by a detector 2520. Asthe wavelength sweeps in time, the A/D converter 2525 digitizes theoutput of the detector 2520 to generate the OCT interferogram 2505.

$\begin{matrix}{{I\left\lbrack k_{m} \right\rbrack} = {\frac{\rho\left\lbrack k_{m} \right\rbrack}{2}{S\left\lbrack k_{m} \right\rbrack}\left( {R_{R} + R_{S} + {2\sqrt{R_{R}R_{S}}{\cos\left( {2{k_{m}\left( {z_{r} - z_{s}} \right)}} \right)}}} \right.}} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

The term inside the cosine function represents the phase of the OCTinterferogram or alternately called OCT fringe. As the phase increases(or decreases), the OCT fringe oscillates with a full period ofoscillation occurring every 2*π radians. A wavelength sweep has astarting wavenumber, k_(start), and an ending wavenumber, k_(end). Thenumber of oscillations in the OCT fringe is proportional to themagnitude of the total phase difference, ΔΦ, over the sweep, which isgiven byΔΦ=2(k _(end) −k _(start))(z _(r) −z _(s)).  Eq. 2

Equation 2 shows that the fringe frequency increases with increasingimaging depth (i.e., there is a larger number of oscillations over thesweep) because the (z_(r)−z_(s)) multiplier term inside the cosinefunction increases total fringe phase. FIG. 26 shows simulatedinterferograms resulting from a stationary mirror reflection in thesample arm with wavelength sweep from a starting wavelength, λ_(star),to an ending wavelength, λ_(end), which given that k=1/λ is equivalentto a starting wavenumber, k_(start), and ending wavenumber, k_(end), andalso where the sample points in the interferogram are equally spaced inwavenumber, as is commonly performed in swept source OCT by opticalclocking or numerical calibration methods. FIG. 26(A), top, shows aninterferogram 2605 from a mirror at a shallow depth and FIG. 26(B),bottom, shows and interferogram 2610 from a deep depth. As expected fromEq. 1 and Eq. 2, the number of oscillations for the deep mirrorreflection is larger than the number of oscillations for the shallowmirror reflection because of the larger overall phase resulting from the(z_(r)−z_(s)) term. FIG. 26(A) shows the interferogram 2615 for astationary mirror with fixed path length in the sample arm. If, in thecase of an embodiment of the present invention, the path length ischanging while the wavelength swept source is sweeping, the effect onthe interferogram is that the beginning portions of the interferogramare associated with the starting optical path length and the endingportions of the interferogram are associated with the ending opticalpath length, which causes the interferogram 2620 to be chirped whencompared to the stationary mirror condition, as shown in FIG. 26(B).FIG. 26(C) shows the OCT point spread function 2625 for the shallowfringe 2605, the OCT point spread function 2635 for the deep fringe2610, and the OCT point spread function 2630 for the chirped fringe2620. FIG. 26(D) shows the OCT point spread function for the shallowfringe 2605 and for a heavily chirped fringe 2645. The chirping has twoeffects on the OCT axial point spread function when Fourier transformed.First, the depth position of the reflection is shifted to some norm oraggregate optical path length position, and second, the point spreadfunction is potentially broadened as it contains information frommultiple depths. Both of these effects are deleterious to OCT imagingperformance. In the case of swept source OCT, sampling rates aregenerally quite fast at several hundred million to 500 million samplesper second (MSPS) or faster with modern A/D cards, resulting in shortsampling times on the order of nanoseconds per sample. The integrationtimes for spectral domain OCT are much longer because the cameras exposemultiple wavelengths and run at tens of kilohertz to several hundreds ofkilohertz rates, resulting in integration times on the order ofmicroseconds, orders of magnitude longer than for swept source OCT. Forspectral domain OCT, an OCT interferogram that changes in time resultsin fringe washout effects that can reduce the fringe contrast andcompromise OCT sensitivity. Fringe washout effects are less pronouncedwith swept source OCT because of the order of magnitude shorterintegrations times. Nevertheless, the current invention can be practicedwith any form of point scanning OCT of image based OCT. Methods foraddressing the optical path length change of the current invention forimproved performance are described next.

Long coherence length swept source lasers enable a long OCT imagingrange to accommodate the change in path length of an embodiment of thepresent invention. Long coherence length swept laser include technologybased on a wavelength tunable vertical-cavity surface-emitting laser(VCSEL), Fourier domain mode locked laser (FDML) and dispersion balancedFDML laser, short cavity laser, and Vernier-tuned distributed Braggreflector (VT-DBR) laser. A long coherence length laser combined with afast detector and high digitization rate enable collection of an OCTimage with sufficient range to accommodate the image distortion createdby the longer optical path length associated with off-axis scanpositions.

A calibration can be applied to the OCT image data (after Fouriertransforming the fringe) that shifts the data in the axial direction inorder to properly align the data in the depth direction to represent thesample morphology. The amount of shift applied to each A-scan can bedetermined by calculation of the nominal path length change expectedfrom the scan geometry or through experimental methods, such as imaginga known flat mirror and determining the required axial shift thatproduces a flat surface in the OCT image data, as shown in FIGS.26(E-F). For many applications, simply shifting the OCT image data issufficient as the slight degradation of OCT axial resolution associatedwith chirping of the OCT interferogram would be acceptable. Forapplications where the highest OCT axial resolution performance isrequired, it can also be beneficial to address the loss of degradationof the OCT PSF associated with the chirping of the OCT interferogram bynumerically correcting the OCT fringe. A proper calibration can beobtained using methods well established and practiced in OCT based onresampling of the OCT fringe and dispersion compensation, such as thosetaught by Section 2.2 of a paper, “Ultra high-speed swept source OCTimaging of the anterior segment of human eye at 200 kHz with adjustableimaging range” by M. Gora, K. Karnowski, M. Szkulmowski, B. Kaluzny, R.Huber, A. Kowalczyk, and M. Wojtkowski, Opt. Express 17, 14880-14894(2009), and Section 2.2 of a paper, “Three-dimensional and high-speedswept-source optical coherence tomography for in vivo investigation ofhuman anterior eye segments” by Y. Yasuno, V. Madjarova, S. Makita, M.Akiba, A. Morosawa, C. Chong, T. Sakai, K. Chan, M. Itoh, and T.Yatagai, Opt. Express 13, 10652-10664 (2005). In these methods, a mirrorreflection or an MZI is used to generate an OCT fringe and numericalmethods applied to create a calibration that is equally spaced inwavenumber, k, and properly dispersion compensated. For an embodiment ofthe present invention, a flat mirror surface can be scanned and acalibration obtained for every A-scan. Storage of the calibration can besimplified by realizing that the perturbation to a nominal calibrationis due to path length change at near constant velocity. So, only thevelocity at any one region in the scan needs to be known, stored, andused to calculate the OCT fringe correction.

An alternate approach to address the change in path length introduced bythe beam projection module is to adjust the optical path length with afast actuator such that the path lengths between the reference arm andthe sample arm remained matched during the scanning. This method wouldbe preferred if performing spectral domain OCT to reduce fringe washouteffects that are more pronounced than in swept source OCT. The pathlength change could be obtained by using a fast and flexible delay lineor by adjusting the path length with a fast actuated mirror. The delayline or fast actuated mirror could be actuated by piezo,electromagnetic, or other actuation. The position of the active mirrorwould be determined by calibration from a flat mirror reflection, bycalculation or simulation such as shown in FIG. 25(F), or other methods.

Other AO System Embodiments

Other embodiments of the present invention are possible. Any of theimaging systems shown in FIG. 1 can be practiced with an embodiment ofthe present invention by replacing the steering mirror shown with a beamprojection module and adaptive optics element. An imaging system forgeneral purpose OCT, similar to that shown in FIG. 1(A), would replacethe steering mirror 135 with a beam projection module and adaptiveoptics element to realize an adaptive optics OCT imaging system of anembodiment of the present invention. An imaging system, similar to thatshown in FIG. 1(B), would replace the steering mirror 155 with a beamprojection module, adaptive optics element, and controller to realize anadaptive optics OCM imaging system of an embodiment of the presentinvention when combined with a suitable interferometer, detector, andemission source. An imaging system, similar to that shown in FIG. 1(C),would replace the steering mirror 172 with a beam projection module, anadaptive optics element to realize an adaptive optics OCT imaging systemfor imaging the eye of an embodiment of the present invention whencombined with a suitable interferometer emission source and detector. Animaging system, similar to that shown in FIG. 1(D), would replace thesteering mirror 177 with a beam projection module, an adaptive opticselement, and controller to realize an adaptive optics confocal imagingsystem of an embodiment of the present invention when combined with asuitable emission source and detector. An imaging system, similar tothat shown in FIG. 1(E), would replace the steering mirror 192 with abeam projection module, an adaptive optics element, and controller torealize an adaptive optics laser scanning ophthalmoscope imaging systemof an embodiment of the present invention when combined with a suitableemission source. An imaging system, similar to that shown in FIG. 1(F),would replace the steering mirror 199 with a beam projection module, anadaptive optics element, and controller to realize an adaptive opticsmultiphoton or second harmonic imaging system of an embodiment of thepresent invention when combined with a suitable emission source. Anoptical tweezers implementation of the current invention can also berealized by replacing the beam steering mirrors in an optical tweezersetup with a beam projection module, an adaptive optics element, andcontroller. In optical tweezers, the detector is often a digital camera.

Conjugation of Adaptive Optics Element

Selecting the plane of conjugation of an adaptive optics element in anoptical system is an important consideration for achieving a largecorrectable field of view with a single adaptive optics correction.Because the light traverses a different optical path as the beam isscanned across the sample, the can aberrations change for each fieldposition. The rate of change of the aberrations and associated pointspread function (PSF) with field position depends on the characteristicsof the aberrating source and details of the optical layout. Theisoplanatic patch is a measure of how quickly the PSF changes with fieldposition and is sometimes defined as the region over which the root meansquare (RMS) wavefront difference between any two wavefronts within thepatch is less than a critical value, although alternate definitions ofthe isoplanatic patch have also been used in the literature. In thispatent application, the concept of instantaneous diffraction limitedfield of view is used to evaluate and compare adaptive opticsperformance, where diffraction limited is defined as a Strehl ratiogreater than or equal to 0.8, and instantaneous indicates using only asingle adaptive optics correction. In certain applications, theimprovement in imaging performance may be significant, but not reachperformance of the diffraction limit. Improvement in the performance insimilar fields of view, or an increase if the size of the field of viewat a critical performance level are also useful and can be achieved withan embodiment of the present invention.

Most adaptive optics imaging system literature and description teachconjugating the adaptive optics element to a pupil plane of the system.In adaptive optics microscope systems, the adaptive optics element isoften conjugated to the pupil plane of the microscope objective, astaught in the papers, “Adaptive optics via pupil segmentation forhigh-resolution imaging in biological tissues” by N. Ji, D. Milkie, andE. Daniel, Nature Methods, 7, 141-147 (2009), a paper, “Image-basedadaptive optics for two-photon microscopy” by D. Débarre, E. Botcherby,T. Watanabe, S. Srinivas, M. Booth, and T. Wilson, Opt. Lett. 34,2495-2497 (2009), and other papers. In adaptive optics scanning laserophthalmoscopes and adaptive optics OCT systems, the adaptive opticselement is often conjugated to the pupil of the eye. For scanningoptical systems, conjugating the adaptive optics element to the pupilmaximizes the number of actuators across the imaging beam and results ina stationary beam center in both the adaptive optics plane and the pupilplane while scanning. A paper, “Requirements for discrete actuator andsegmented wavefront correctors for aberration compensation in two largepopulations of human eyes” by N. Doble, D. Miller, G. Yoon, and D.Williams, Appl. Opt. 46, 4501-4514 (2007), investigates the requirementson the stroke and number of actuators across the pupil in populations ofhuman eyes. A different paper, “Statistical variation of aberrationstructure and image quality in a normal population of healthy eyes” byL. Thibos, X. Hong, A. Bradley, and X. Cheng, J. Opt. Soc. Am. A 19,2329-2348 (2002), investigates the type and magnitude of aberrations ina normal population of eyes. In both of these papers, wavefrontmeasurements are performed along the line of sight, so only a singlefield position on the eye is investigated. A practical adaptive opticsimaging system images not only at a single point, but over an extendedfield of view. It is also desirable that the adaptive optics correctionapply to as large a field as possible. The optimal adaptive optics pupilconjugation can be determined through simulation or experimentalmethods. The effects of adaptive optics element conjugation aredemonstrated next through optical simulation.

The Liou and Brennan model of the eye is one of the most accurate modelsof the human eye developed to date and includes morphologically similarsurface contours to the eye, gradient index refractive properties of thelens, and an offset pupil position similar to the eye. The model hasbeen shown to match physiologically obtained experimental data, asdescribed in a paper, “Different Schematic Eyes and their Accuracy tothe in vivo Eye: A Quantitative Comparison Study” by MS de Almeida andLA Carvalho, Brazilian Journal of Physics 37, pp. 378-387 (2007). FIG.27(A) shows a ZEMAX simulated ray trace of a model of the human eye 2705based on the Liou and Brennan model, but with the pupil centered withrespect to the optical axis. The pupil diameter is 4 mm, which is largerthan the diameter expected to produce optimal lateral imaging resolutionbecause of residual aberration.

Studies of the aging process of the eye have found that the primarycause of increasing refractive error with age is due to changes in thecrystalline lens rather than changes in the cornea, as described in apaper, “Optical aberrations and alignment of the eye with age” by EstherBerrio, Juan Tabernero, Pablo Artal, Journal of Vision 10(14) (2010). Ina normal and young eye, the aberrations of the cornea 2710 are balancedby aberrations in the crystalline lens 2715. Thus, the source of theaberration in a normally aging eye is not located at the pupil planeitself, but originate because of an imbalance of aberration between thecornea 2710 and crystalline lens 2715. FIG. 27(B) shows the traditionaladaptive optics design of a deformable mirror 2720 conjugated to thepupil plane of the eye using a 4f telescope, where the telescope iscomposed of a first 2725 and a second 2730 paraxial lens surface inZEMAX. The paraxial lens surfaces act like ideal lenses and do notintroduce any aberration of their own. A surface capable of introducingphase error (Zernike fringe phase surface) is located immediatelyfollowing the crystalline lens 2735 and acts to create an additionalimbalance of aberration between the cornea and back surface of thecrystalline lens. Light from the deformable mirror 2720 in thissimulation is planar and aberration free. Collimated light from thedeformable mirror 2720 propagates to the first lens 2725 of the afocaltelescope system where it is focused to a converging beam that is inturn collected by the second lens 2730 and re-collimated for projectioninto the eye model 2705. Aberration free light enters the eye model2705, is subject to the aberrations inherent in the eye, additionallysubject to the aberrations introduced by the aberrating surface, andfocuses on the retina. The aberrating surface 2735 is configured togenerate astigmatism phase error 2740. A wavefront analysis 2745 of thelight at the retina shows the dominate shape of the aberration sourcecombined with the eye's natural aberration.

FIG. 28 shows additional information about this traditional approach ofconjugating the deformable mirror to the pupil in the eye. FIG. 28(A)shows the conjugate image planes in the system. FIG. 28(B) shows theconjugate pupil planes in the system, where it can be seen that thedeformable mirror is imaged to the pupil plane of the eye. FIG. 28(C)shows zoomed in ray traces of the deformable mirror and the crystallinelens in the eye where the chief ray, the marginal rays, and an auxiliaryray are labeled. As expected, the chief ray and auxiliary ray are imagedfrom the deformable mirror to the pupil of the eye with relativeordering and normalized spacing with the beam diameter preserved.However, at the plane of the source of the aberration, the chief ray andthe auxiliary ray overlap and cross. This means that a correctionapplied at the deformable mirror will be blurred at the plane of thesource of the aberration and consequently less effective at compensatingfor the aberrations introduced at this plane.

FIG. 29 shows an alternate design in which the lens spacing of thetelescope has been adjusted so that the deformable mirror is nowconjugated to a plane that is approximately located at the plane of thesource of aberration. FIG. 29(A) shows a ray trace of the conjugateimage planes. FIG. 29(B) shows a ray trace of the conjugate pupilplanes, in which the conjugation between the deformable mirror and theapproximate plane of the source of aberration can be seen. FIG. 29(C)shows zoomed in ray traces of the chief ray, marginal rays, and anauxiliary ray. In this alternate configuration, it can be seen that thechief ray and the auxiliary ray intersect at the deformable mirror andalso intersect at the plane of the source of aberration. This means thata corrective shape at the deformable mirror is spatially localized inthe plane of the source of aberration and is effective at cancelling anaberration at more than one field position.

Imaging over a field size of 4.5 degrees by 4.5 degrees, FIG. 30compares the performance of the pupil conjugated configuration shown inFIG. 28 and the aberration source conjugated configuration shown in FIG.29. In these simulations, the deformable mirror (Zernike fringe phase)was parameterized with Zernike modes 4-27 and ZEMAX optimization wasused to optimize the deformable mirror shape to simultaneously minimizethe RMS wavefront error over five field positions of (0,0), (0,4.5),(2.25, 2.25), (4.5,0), and (4.5,4.5). In the case of the pupilconjugated configuration, the position of the deformable mirror wasfixed in order to maintain pupil conjugation. In the case of theaberration conjugated configuration the position of the deformablemirror relative to the first lens of the afocal telescope was defined asa variable and allowed to change during the optimization. In the case ofthe aberration conjugated configuration, the active diameter of thedeformable mirror was forced to match the position of the most extremeray in the (4.5, 4.5) field position. By using the same Zernike terms inthe two different configurations, the relative spatial frequencycorrecting capability of the deformable mirrors are the same for faircomparison, i.e., the actuator count and influence function of the twomirrors are identical when normalized to the diameter of the adaptiveoptics element. The results show that over the same field size, theaberration source conjugated configuration outperforms the pupilconjugated configuration with respect to Strehl ratio at all fieldpositions. The aberration source conjugated configuration hasdiffraction limited performance (Strehl ratio greater than 0.8) over theentire 4.5 by 4.5 field of view, while the pupil conjugated system isnot diffraction limited over the entire field of view. This implies thatwhen the aberrations in the eye are primarily due to refractive errornear the back surface of the crystalline lens, the optimal position ofthe adaptive optics element is outside of the pupil plane and somewherebetween the pupil plane and the plane of aberration shown. Note that inorder for this out-of-pupil scanning scheme to work, the beam diametermust be smaller than the active diameter of the deformable mirror andthe beam center must move on the deformable mirror surface, as shown inFIG. 31(E). This results in a small loss of actuator density across thebeam, but shows that the advantage of conjugating to the source ofaberration outweighs the small loss of actuator density.

In the eye, it is possible to locate the general region of the source ofthe aberration and conjugate to, or approximately conjugate the adaptiveoptics element to the source of aberration because the source of theaberration is located close to the pupil plane and there is sufficientdistance between source of the aberration and the focal plane. In amicroscope based imaging system, the source of the aberration isgenerally very close to the focal plane and is due to the sample itselfor a material or optical interface that is adjacent to, touching, ornear the sample and imaging plane. It is therefore not necessarilypossible to completely conjugate the adaptive optics element to thesource of aberration in a microscope system. FIG. 31 (A-C) show amicroscope objective where the design form for the lens is derived froma lens prescription described in U.S. Pat. No. 6,501,603. A zoomed viewof the rays focusing into the sample as emerging from the last lens inthe microscope objective is shown in FIG. 31(A). The objective is awater emersion type and a ZEMAX simulation of the optical performance ofthe objective contains a layer of water following the last glass elementof the objective. The layer of water is then followed by alternatingZernike Phase surfaces and thin water layers before a final layer ofwater in which there is the optical focus. The Zernike Phase surfacessimulate the more realistic effect of sample induced optical aberrationoccurring through a depth of the sample (i.e. the aberrations are notcontained in a single plane). The shapes of the phase errors introducedby the Zernike Phase surfaces are shown in FIG. 31(A) and togetherintroduce about 1 wave of peak-to-valley aberration. The ability of thedeformable mirror to correct the aberrations introduced by the sample iscompared by defining the deformable mirror with a Zernike Phase surfaceusing Zernike terms 4-20 and optimizing the shape of the phasecorrection of the deformable mirror to simultaneously minimize the rootmean square (RMS) wavefront error over field positions of 0.0 degrees,2.5 degrees, and 5.0 degrees input angles. A configuration in which theadaptive optics element is conjugated to the pupil of the objective,shown in FIG. 31(B), is compared to a configuration in which theadaptive optics element is shifted away from the pupil plane by 15 mm,shown in FIG. 31(C). The adaptive optics element is conjugated to aregion in the microscope objective by a telescope composed of paraxiallens surfaces with focal lengths of 50 mm. The optimal adaptive opticscorrections are shown in FIGS. 31(B) for the pupil conjugatedconfiguration and 31(C) for the out-of-pupil conjugated adaptive opticsconfiguration. The resulting Strehl ratios for the field positions of0.0, 2.5, and 5.0 degrees are 0.758, 0.887, 0.702 for the pupilconjugated configuration and 0.805, 0.926, 0.837 for the out-of-pupilconjugated configuration. Over the same field size, the out-of-pupilconjugated configuration outperforms the pupil conjugated configuration,indicating the larger diffraction limited field of view obtained withthe out-of-pupil conjugated configuration.

Larger diffraction limited fields of view have been demonstrated without-of-pupil conjugation of the adaptive optics element with examplesand simulations of imaging in the human eye and in two-photon imagingthrough an aberration generating sample over extended field sizes.However, it is important to realize that placing the adaptive opticselement out of the pupil plane reduces the effective number of actuatorsacross the beam. FIG. 31(D) shows a beam size that is equal to theactive area of the adaptive optics element and thus maximizing theactuator count across the beam, as is achieved when conjugating theadaptive optics element to the pupil plane of the system. In the case ofplacing the adaptive optics element outside the pupil plane, the beamsize must be smaller than the active area of the adaptive optics elementas the beam center position must move as a function of the scan angle,as shown in FIG. 31(E). For a given adaptive optics element, thisnecessarily reduces the actuator count across the beam, whichpotentially adversely affects wavefront correction. For large fieldsizes, the advantages offered by placing the adaptive optics elementoutside the pupil plane of improved correction over a larger field ofview outweigh the disadvantage of lower spatial frequency correctionover the beam. As the size of the desired field of view decreases, themagnitude of the change in wavefront over the field of view alsodecreases and the effects of anisoplanatism become less pronounced suchthat increasing the number of actuators across the beam can be morebeneficial to performance than moving the adaptive optics elementoutside of the pupil plane and closer to conjugation to the source ofthe aberration. In the limit of a single point field of view, thewavefront does not change at all over the field of view and the bestadaptive optics performance will likely be obtained by maximizing thenumber of actuators across the beam by conjugating the adaptive opticsto a pupil plane of the system. In one embodiment of the presentinvention, the adaptive optics element 715 is conjugated to a pupilplane of the system. In another embodiment of the present invention, theadaptive optics element 715 is conjugated to a plane outside of thepupil plane to improve adaptive optics correction. Improved adaptiveoptics correction would constitute imaging to a particular Strehl ratioover an enlarged field of view, or imaging over a similar sized field ofview, but with improved Strehl ratio within the field of view. As can beseen in FIG. 28(C), the beam pivot point is located at the adaptiveoptics element. As can be seen in FIG. 29(C) and FIG. 31(C), the beampivot point is located near, but not at the adaptive optics element. Itis in this sense of near that the beam projection module operates withfour or more axes of motion and controls an angle and position of thelight to preferentially interface the adaptive optics element(s) bycreating or accommodating a beam pivot point at or near the adaptiveoptics element(s).

Ordering of Adaptive Optics Element

It is possible to change the ordering of the beam projection module andadaptive optics while still preserving the essential functionality. Inone embodiment, the beam projection module 3215 is located before theadaptive optics element(s) 3205 in the system, as shown in FIG. 32(A).This embodiment is generally preferred because the conjugation betweenthe adaptive optics element 3205 and the sample delivery optics 3210 isnot affected by path length change within the beam projection module3215. In another embodiment, the adaptive optics element(s) 3220 in theoptical system is located before the beam projection module 3225, asshown in FIG. 32(B). In this configuration, small changes in the opticalpath length of the beam projection module 3225 may cause a positiondependent axial shift in the plane that is conjugate to the adaptiveoptics element. Another embodiment splits the axes of the beamprojection module such that a group of axes 3235 is located before theadaptive optics element 3240 and a group of axes 3245 is located afterthe adaptive optics element 3240. This configuration also suffers from achange in optical path length which affects the conjugation between theadaptive optics element and the intended plane of conjugation. In oneembodiment of the adaptive optics scanning system, the beam projectionmodule 720 directs light to the adaptive optics element(s) such that acenter of the light beam remains predominately aligned with a center ofthe adaptive optics element(s) 715 while the angle of light beamrelative to the adaptive optics element(s) 715 is changed during a beamsteering operation. In another embodiment of the adaptive opticsscanning system, the beam projection module 720 receives light from theadaptive optics element(s) 715 and directs the light such that a centerof the light beam remains predominately aligned with a center of adesired pupil plane in the imaging system while the angle of light beamrelative to the desired pupil plane is changed during a beam steeringoperation.

Focus and Conjugation Control

It is common in microscopy that the objective be able to translate toaccommodate different specimen heights and sizes, as well as to focus toa plane of interest in the sample. One embodiment of comprises a meansfor adjusting the focus in the sample, as shown in FIG. 33. Morespecifically, an embodiment of the present invention includes the casewhere the imaging system comprises a means for adjusting the focus bytranslating a microscope objective, scan lens, or objective lens as partof the sample delivery optics. When the objective location changes, itis still desirable to maintain beam alignment and pupil conjugation withthe adaptive optics element. An embodiment of the present inventionincludes the case where the motion trajectories of the controller changeto accommodate changes in focus while maintaining proper alignment ofthe light beam with the pupil of the sample delivery optics. Anembodiment of the present invention also includes the case where opticalelements within the sample delivery optics move to accommodate changesin focus while maintaining proper alignment of the light beam with thepupil of the sample delivery optics. It is also possible to affect focuswithout moving the position of any of the optical elements. Anembodiment of the present invention includes the case where a defocusmode is generated with the adaptive optics to achieve focus positioncontrol within the sample.

For a variety of reasons related to resolution, field of view, depth offield, and other, it may be desirable to change the sample deliveryoptics or objective. An embodiment of the present invention includes thecase where different objectives can be accommodated that have differentpupil positions by adjusting the scan trajectories in the beamprojection module, by adjusting or changing optical elements in thesample delivery optics, or adjusting both scan trajectories in the beamprojection module and optical elements in the sample delivery optics.

By monitoring light coming out of the objective while scanning, it ispossible to infer and assess the quality of optical alignment. Anembodiment of the present invention includes the case where acalibration is performed with the objective in place to learn the pupilposition of the objective. Further, an embodiment of the presentinvention includes the case where elements in the sample delivery opticsare changeable or adjustable to accommodate different objective pupildiameters, different objective pupil locations, or both differentobjective pupil diameters and pupil locations. In one possibleimplementation, a zoom beam expander is used in the sample deliveryoptics to accommodate different pupil sizes.

Optional Enhancements and Alternative Embodiments

Different adaptive optics technologies and designs have differentperformance characteristics.

In one embodiment, the number of adaptive optics elements is two or moreand a combination of adaptive optics elements is used to increase therange of wavefront correction, intensity correction, or both wavefrontand intensity correction. In one embodiment, the number of adaptiveoptics elements is two or more and the two or more adaptive opticselements have different correction range, actuator or pixel arrangement,actuator or pixel spacing, or temporal response to achieve a correctionthat is preferred over using any one of the adaptive optics elementalone. In one embodiment, two or more adaptive optics elements are usedin a woofer-tweeter configuration, as shown in FIG. 34. A 4f telescopemay be used between two adaptive optics elements or the two adaptiveoptics elements may by located in close proximity to each other. In oneembodiment, a liquid crystal spatial light modulator is mounted near thereflective surface of a deformable mirror. This arrangement can bedesirable because it allows the deformable mirror and liquid crystalspatial light modulator to be conjugated to nearly the same plane. Inone embodiment, a liquid crystal spatial light modulator corrects largeamplitude aberrations, but is limited to slow dynamic performance, whilethe deformable mirror corrects smaller amplitude aberrations, butoperates with fast dynamic performance. In another embodiment of thepresent invention, two or more beam projection modules 720 are used tocascade multiple adaptive optics element(s) 715, each beam projectionmodule 720 operating with four or more axes of motion.

For applications where the optical performance is affected bydispersion, an embodiment of the present invention can include adispersion compensation unit to compensate for dispersion in the system.One embodiment of the present invention includes a dispersioncompensation unit, the dispersion compensation unit being comprised ofany one or more of the following: dispersion compensating mirrors (DCM),prisms, glass wedges, gratings, or active dispersion compensation bymeans of an active deformable mirror or spatial light modulator.

It is possible to perform parallel imaging with two beams in certainimaging modalities. The two beams may originate from two closely spacedfiber optic fiber tips or from two beams with different propagationangles. An embodiment of the present invention includes the case wheremultiple beams pass through the imaging system to perform parallel spotimaging.

The method of beam steering with the beam projection module 720 has beenshown with collimated beams. However the same method works forconverging or diverging beams as long as the beam stays within themirror limits. One embodiment of the present invention uses convergingor diverging beams in the beam projection module 720.

During setup and alignment of the adaptive optics scanning system, thebeam from the emission source often needs to be precisely aligned withthe intended optical axis of the optical system. Alignment may driftover time and temperature. It is possible to determine the quality ofalignment by monitoring the beam position and using a sensor, as shownin FIG. 35. An embodiment of the present invention includes the casewhere one or more position sensing or angle sensing detector(s) is usedto determine the accuracy of incoming beam alignment to the beamprojection module from the emission source and information used aboutthe beam alignment to correct for misalignment by adjusting the scantrajectories of the active axes. Further, the sensor for monitor thebeam position and alignment may be included in the optical path bychanging one or more of the active mirrors in the beam projection moduleto direct light from the normal imaging path to the alignment detector,as shown in FIG. 35. One embodiment of the present invention includesone or more 1D or 2D detector(s), such as CCD array, CMOS array, orposition sensing diode (PSD), or any other detector that can measure abeam position is used to monitor the beam position with or without asmall beam splitter or additional mirror to check the quality of beamalignment.

Adaptive Optics Control

In adaptive optics systems, it is common that light from a point source(guide star) be used to estimate the optical aberrations. One embodimentof the present invention includes a wavefront sensor for measuring anaberration in the light from the sample or a point source within thesample. In this embodiment, the imaging system may determine anappropriate adaptive optics correction by using information about theaberration obtained with the wavefront sensor, as shown in FIG. 36(A).When using a wavefront sensor, an algorithm for adjusting the deformablemirror that is commonly used in practice is to execute the steps ofmeasuring the wavefront 3605, calculating an adaptive optics correction3610, and applying the correction to the adaptive optics element 3615.Most adaptive optics systems place the wavefront sensor before thescanners so that the beam entering the wavefront sensor is collinearwith the excitation beam being directed to the sample. A dichroic mirroror beam splitter and a light source for the beacon would be locatedbetween the emission source 705 and beam projection module 720 of anembodiment of the present invention to generate the guide star for thewavefront sensor, as taught in the before mentioned Dubra 2011 paper.Alternatively, the wavefront sensor could be located after the beamprojection module 720 and a dichroic mirror or beamsplitter includedwith appropriate pupil relay as part of the sample delivery optics 730.The advantage of locating the wavefront sensor after the beam projectionmodule is that the conjugate of the wavefront sensor to the pupil doesnot change with beam steering position, however, the excitation beammust be precisely centered so as to not introduce significant tilt modesinto the wavefront sensor measurement. The advantage of locating thewavefront sensor before the beam projection module are that tilt modesare not introduced while scanning, however there may be a small pathlength change during scanning that affects the conjugation of thewavefront sensor to the plane of conjugation.

Other methods exist for determining the proper adaptive opticscorrection. One technique, often referred to as wavefront sensorlessadaptive optics, optimizes the adaptive optics component usinginformation from the image or sample signal alone. Papers that teachalgorithms for sensorless adaptive optics include “Image based adaptiveoptics through optimisation of low spatial frequencies” by D. Debarre,M. Booth, and T. Wilson, Opt. Express 15, 8176-8190 (2007), “Image-basedadaptive optics for two-photon microscopy” by D. Débarre, E. Botcherby,T. Watanabe, S. Srinivas, M. Booth, and T. Wilson, Opt. Lett. 34,2495-2497 (2009), and others “Adaptive optics via pupil segmentation forhigh-resolution imaging in biological tissues” by N. Ji, D. Milkie, andE. Daniel, Nature Methods, 7, 141-147 (2009). In wavefront sensorlessadaptive optics control, there is often an iterative loop of perturbingthe adaptive optics element to obtain input/output data between theadaptive optics element and the signal, as shown in FIG. 36(B). Theinner loop consists of steps of applying shapes (basis functions, orsometimes called modes) to the adaptive optics element 3620 andmeasuring and storing the signal response 3625. The results of the innerloop are used to calculate an adaptive optics correction 3630, which isthen followed by applying the correction to the adaptive optics element3635. One embodiment of the present invention determines an appropriateadaptive optics correction by using a wavefront sensorless adaptiveoptics optimization method. Many wavefront sensorless methods apply aseries of shapes or alternatively called basis functions, or modes tothe adaptive optics element as part of the optimization process. Thequality of correction can be assessed by calculating a metric associatedwith a measurement of the light returning from the sample with thedetector. An embodiment of the present invention includes the case wherethe adaptive optics optimization methods generate a series of adaptiveoptics shapes, applies the shapes to the imaging system, assesses theimpact of the shapes by calculating a metric value based on measurementsof the light from the detector, and updates the adaptive optics elementto improve image or signal quality. The metric is usually a measure ofsignal quality, contrast, or spatial frequency content, as taught in thebefore mentioned papers (Debarre, 2007, Debarre 2009, Ji, 2009). Theoptimization algorithm can be any one of many optimization algorithmsknow in the field of optimization, including Newton's method,quasi-Newton methods, gradient descent, conjugate gradient, geneticalgorithms, simulated annealing, hill climbing, polynomialinterpolation, or other optimization algorithm known in the art ofnumerical optimization Optimization of the adaptive optics can beperformed by zonal or modal control methods. In zonal methods, localregions of the adaptive optics actuators or pixels are controlledseparately. In modal control methods, multiple actuators or pixels arecontrolled simultaneously with a set of basis shapes. When using modaltechniques, one embodiment of the present invention uses profiles of theadaptive optics mode shapes that are predominately orthogonal to improvethe rate of convergence of an optimization algorithm. Certain modes ofaberration correction do not improve the image quality. For example,piston changes the absolute phase of the wavefront, but not theresulting point spread function (PSF). Tip and tilt steer the beam, butdo not affect the image quality. It is therefore sometimes desirable toremove piston, tip, and tilt from the modes controlling the adaptiveoptics element. An embodiment of the present invention includes the casewhere the profiles of the adaptive optics shapes are generated to avoidincluding portions of piston, tip, and tilt modes. In somecircumstances, certain mode shapes are more important than others.Optimization can be performed on a subset of modes, as shown in FIG. 37.Only three basis shapes (modes) are used in the optimization, as shownby the plot 3705 showing the basis amplitudes. The convergence plot 3710shows the progress of the optimization algorithm. An image of the samplewith the deformable mirror flat 3715 is compared to an image of thesample with the deformable mirror optimized 3720. The image of thesample with the deformable mirror optimized 3720 shows increased signaland improved resolution when compared to the image of the sample withthe deformable mirror flat 3715.

Many imaging modalities are depth sectioning imaging modalities, such asconfocal, multiphoton, and others. For sectioning imaging modalities, itis desirable to correct image degrading aberrations at a particularfocal depth in the sample. In this case, it is desirable to remove anydefocus mode from the basis set controlling the adaptive optics element.An embodiment of the present invention includes the case where theprofiles of the adaptive optics corrections are generated to avoidincluding portions of defocus modes. Given an optimized adaptive opticsstate for a particular region in a sample, it is likely that regionsnearby will have similar aberrations. It is therefore possible toinitialize the adaptive optics with a state for a nearby region todecrease the time required to achieve convergence. Information about anappropriate adaptive optics correction from more than one region can becombined with the goal of improving the estimate for a new region in thesample. An embodiment of the present invention includes the case whereinformation about an appropriate adaptive optics correction for a firstlocation or multiple locations within the sample is used to estimate anappropriate adaptive optics correction for a new location within thesample.

When performing OCT imaging, frequency and phase information containedwithin the OCT fringe contain information about the path length of lightcoming from the sample. The information encoded in the OCT fringe can beused to estimate a wavefront. In confocal or multi-photon imaging,methods such as blind deconvolution can be used to estimate a pointspread function, an object, and a wavefront. One embodiment of thepresent invention uses a wavefront estimated from OCT data or from imageprocessing methods, such as blind deconvolution, as part of theoptimization processes for determining a correction for the adaptiveoptics element 715.

In optical tweezer systems, the adaptive optics can be optimized usingalgorithms such as those taught in a paper, “Holographic opticaltweezers aberration correction using adaptive optics without a wavefrontsensor” by K D. Wulff, D G. Cole, R L. Clark, R D Leonardo, J Leach, JCooper, G Gibson, M J Padgett, Proc. SPIE 6326, Optical Trapping andOptical Micromanipulation III, 63262Y (2006) and “Combinedholographic-mechanical optical tweezers: Construction, optimization, andcalibration”, by R D L Hanes, M C Jenkins, and SU. Egelhaaf, Rev. Sci.Instrum. 80, 083703 (2009).

Beam Switching

FIG. 38(A) shows a diagram of a beam projection module viewed along thex axis and FIG. 38(B) shows a diagram of the same beam projection moduleviewed along the y axis. Three different input beams 3805, 3810, and3815 are aimed such that they cross at a point that is coincident with asteering mirror 3820. The angle between the incoming beams is smallenough that rotation of the steering mirror 3820 enables selecting whichof the input beams 3805, 3810, or 3815 is passed through the opticalsystem. If the angle between the incoming beams 3805, 3810, and 3815 istoo small, then it is possible that an unintended portion of an inactivebeam also pass through the optical system. Unintended transmission of abeam through the optical system can be prevented by ensuring that theangle between the beams is large enough that the distance between beamedges at the mirror 3825 is larger than the mirror surface. Unintendedtransmission of a beam through the optical system can be prevented byensuring that the angle between the beams is large enough that a fieldstop within the optical system blocks transmission of the unintendedbeam. Capability of switching between input beams could be desirablewhen performing multiple modes of imaging. A single instrument canperform different imaging modalities by switching between emissionsources and other related systems. For example, a combined two-photonand OCT imaging system might use a Titanium Sapphire laser centeredaround 850 nm and an OCT system centered around 850 nm, 1050 nm, or 1310nm. Light from the Titanium Sapphire laser 3830 generates a beam 3835that is directed to the beam projection module 3840. Light from the OCTsystem is delivered by a fiber optics cable 3845 and collimated into abeam 3850 that is also directed into the beam projection module 3840.The beam projection module enables switching between the two input beamsto direct the light through sample delivery optics 3855 to a sample3860. In the mode of two-photon imaging, excitation light passes througha long pass filter 3865, while fluorescent emission light from thesample reflects off the long pass filter 3865 and is directed to a PMTdetector 3870. In the mode of OCT imaging, light centered around 850 nm,1050 nm, or 1310 nm passes through the long pass filter 3860 in thedirection towards the sample and also passes backscattered and reflectedlight from the sample 3860 through the long pass filter and back throughthe beam projection module to the OCT interferometer. Using a multimodalimaging system, additional information can be gathered about the sampleand equipment can be timeshared for different imaging modalities in acompact installation.

Modular Adaptive Optics Unit

The basic concept of the beam steering module and adaptive opticselement as previously described can be considered as a modular adaptiveoptics unit for adaptation to other instruments. The modular adaptiveoptics unit could be sold as a stand alone module for the user tointegrate with their own optical system, as an original equipmentmanufacturer (OEM) module, or as part of an integrated system. Oneembodiment of the beam steering module and adaptive optics elementportion of a modular adaptive optics unit are shown in FIGS. 13-15. Asshown in FIG. 38(D), one embodiment of the modular adaptive optics unitis comprised of one or more entrance ports, the entrance ports allowingone or more optical beams to enter the modular adaptive optics unit asshown in FIG. 38(A), one or more output ports, the output ports beinglocated along one or more beam paths at which the optical beam maytransit or be terminated, one or more adaptive optics element(s), theadaptive optics element(s) affecting the wavefront, affecting theintensity, or affecting both the wavefront and intensity of the lightbeam, a set of beam steering elements, the beam steering elementscreating four or more axes of motion that affect the angle of, and/orthe transverse position of, the propagation path of the light topreferentially create at least one effective rotation point about whichthe light beam is pivoted, and a means for controlling the trajectoriesof the beam steering elements to direct the light beam alongpreferential paths. The entrance ports and output ports may be physicalports or simply different optical paths. The means for controlling thetrajectories include all means for controlling the trajectoriespreviously discussed for the controller 725.

In FIG. 38(D), an embodiment of a modular adaptive optics unit 3875receives light from a first beam 3880 and a second beam 3885 and directslight to an optical subsystem 3887.

In an optical system one may wish to condition light beams or to protectcertain optical elements from contamination or by limiting access tothese certain elements. To achieve this goal one or more of the entranceports 3890 and output ports 3895 of one embodiment of the modularadaptive optics unit 3875 contain any combination of the following:optical window, an optical filter, a band-pass filter, a notch filter, along-pass filter, a short-pass filter. This list is not to be considereda complete list of possible optical elements that may be used in theseports; but, it is a sampling of common elements that may be used. Theseelements may be fixed or removable. In one embodiment of the modularadaptive optics unit, one or more optical filters are removable.

The adaptive optics element of one embodiment of the modular adaptiveoptics unit 3875 may include one or more deformable mirrors. Oneembodiment of a modular adaptive optics unit comprises an adaptiveoptics element(s) that is a deformable mirror. Deformable mirrors of anembodiment of the modular adaptive optics unit may comprise a continuousfacesheet or a segmented facesheet, electrostatic actuators,piezo-electric actuators, unimorph piezo actuators, bimorph piezoactuators, pneumatic actuators, or other equivalent means to deform thefacesheet. Examples of these deformable mirror elements are shown inFIG. 3. Deformable mirrors in one embodiment of the modular adaptiveoptics unit may be of MEMs type structure, membrane type structure,layered piezo type structure, tip/tilt/piston or tip/tilt element typestructure, or other type structure able to repeatedly change the shapeof, orientation of, or shape and orientation of the mirror surface.

The adaptive optics element of an embodiment of the modular adaptiveoptics unit 3875 may include one or more spatial light modulators. Oneembodiment of the modular adaptive optics unit uses an adaptive opticselement that is a spatial light modulator. Spatial light modulators maybe based on liquid crystal elements or other methods to modulate theintensity, modulate the phase, or modulate both the phase and intensity.Examples are show in FIG. 3. Spatial light modulators may be used tocompensate for wavefront aberrations or intensity variations caused tothe optical beam before or after the adaptive optics element. Wavefrontand intensity of a beam propagating through an optical system may beaffected by medium through which it travels. These medium include, butis not limited to, gas, liquid, optical windows, glass elements, tissue,filters, lenses, mirrors, diffractive optical elements, active orpassive crystals. One embodiment of the modular adaptive optics unituses an adaptive optics element(s) to compensate for wavefrontaberrations, or intensity variations, or wavefront aberrations andintensity variations, caused to the optical beam by propagating throughan optical medium or optical elements that comprise gas, liquid, opticalwindows, glass elements, tissue, filters, lenses, mirrors, diffractiveoptical elements, active or passive crystals, after transmitting towardand through at least one output port 3895.

Depending on the amount of wavefront or intensity variations in anoptical beam, two or adaptive optics elements may be used to increasethe magnitude of these variations that may be corrected. The adaptiveoptics elements may or may not be substantially similar to each other.They may be used to statically compensate for the variations orcompensation may be varied temporally. For example, in one embodiment ofthe modular adaptive optics unit, two or more adaptive optics elementswith different designs may be used such that the two or more adaptiveoptics elements have different correction range, or actuatorarrangement, or spacing, or temporal response, or any combination ofthese parameters to achieve a correction that is preferred over usingone adaptive optics element alone.

Many adaptive optics systems use optical relays as shown in FIG. 6 toproperly manage the requirements of beam pivot locations in the opticalsystem. The modular adaptive optics unit includes a beam projectionmodule for generating a pivot location for the beam at an appropriatelocation in the optical system. The beam projection module has four ormore axes of motion that affect mirrors to properly guide the beam. Oneembodiment of the modular adaptive optics unit includes the case wherethe axes of motion comprise at least one rotational axis. One modularadaptive optics unit embodiment includes the case where the axes ofmotion comprise at least one translational axis. One modular adaptiveoptics unit includes the case where the axes of motion comprise acombination of rotational and translational axes. One embodiment of themodular adaptive optics unit uses beam steering elements comprising atleast one galvanometer driven mirror. One embodiment of the modularadaptive optics unit of uses beam steering elements comprising fourgalvanometer driven mirrors. One embodiment of the modular adaptiveoptics unit uses beam steering elements comprising at least one faststeering mirror, the fast steering mirror having two axes of rotation.One embodiment of the modular adaptive optics unit uses beam steeringelements comprising two fast steering mirrors, the two fast steeringmirrors having two axes of rotation. One embodiment of the modularadaptive optics unit that uses beam steering elements comprising atleast one resonant scanning mirror. One embodiment of the modularadaptive optics unit using beam steering elements comprising singly orin any combination of the following: a steering mirror, acousto-opticdeflector, rotating polygon, electro-optic beam deflector, electro-opticprism, thermo-optic prism, or diffractive array.

One embodiment of the modular adaptive optics unit operates with thecoordination between the multiple axes of motion controlled throughelectronic signals to the actuators or active elements. One embodimentof the modular adaptive optics unit embodiment operates with thecoordination between these axes of motion controlled through amechanical linkage. Trajectories along which one may wish to control theaxes of motion to direct the beam of light are varied. One embodiment ofthe modular adaptive optics unit embodiment operates with a means forcontrolling the trajectories of the axes of motion changing the path ofthe light beam so that it traces a raster scan pattern in at least oneoutput port, or at a defined plane in an optical system that receivesthe light beam through at least one output port. One embodiment of themodular adaptive optics unit uses beam steering elements to direct thelight beam to the adaptive optics element(s) such that a center of thelight beam remains predominately aligned with a center of the adaptiveoptics element(s) while the angle of incidence of light beam relative tothe adaptive optics element is varied by the means for controlling thetrajectories of the axes of motion. One embodiment of the modularadaptive optics unit uses beam steering elements to receive light fromthe adaptive optics element(s) and direct the light beam such that anapparent center of rotation of the light beam remains predominatelyaligned relative to a point located in a defined plane while the angleof light beam is varied by the trajectories of the axes of motion,wherein the defined plane is located along a beam path after the beamsteering elements.

As mentioned earlier, multiple adaptive optics elements may be requiredto compensate variations in wavefront or intensity or both wavefront andintensity. One modular adaptive optics unit embodiment includes the casewhere two or more beam projection modules are used to cascade multipleadaptive optics elements, such that each beam projection module operateswith four or more axes of motion.

One modular adaptive optics unit embodiment of the adaptive opticsscanning system includes the case where a 4f optical relay is used tomatch the active area of the integrated adaptive optics element to anoptical system receiving the light beam from adaptive scanning system.One modular adaptive optics unit embodiment includes the case that a 4foptical relay is used to relay the wavefront incident on the adaptiveoptical element to a conjugate plane before, substantially at, or afterat least one said exit port to enable interfacing said adaptive opticsscanning system to an optical system receiving said light beam from atleast one said output port. FIG. 6 shows a typical 4f relay that wouldbe used to relay a wavefront from one plane to another.

One modular adaptive optics unit embodiment includes the case where the4f optical relay comprises reflective optical elements, refractiveoptical elements, or a combination of reflective and refractive opticalelements. One modular adaptive optics unit embodiment includes the casewhere the 4f optical relay has variable magnification. One modularadaptive optics unit embodiment includes the case where one or more 4foptical relays may be used to interface the adaptive optics scanningsystem to an optical system receiving the light beam from at least oneoutput port of the adaptive optics scanning system, where the 4f opticalrelay helps to overcome space constraint related to a short distancebetween a pupil plane in the optical system and the first opticalelement of the adaptive optics scanning system.

Many scanning laser systems are used in applications that require pulsedlasers as the light source. Short pulse lasers provide the ability toinput short bursts of optical energy into a system with relatively highpeak powers, where the optical wavelength range is substantially broaderthan a CW laser and is centered at or near a desired wave length. Thewavelength spectrum emitted by pulsed lasers may be tailored, withincertain operating parameters' limits, to the application. If theapplication requires pulses durations at the beam termination point tobe substantially near a certain value, dispersion compensating elementsor systems may be required to compensate for the deleterious affectoptical materials have on the optical pulse duration, and thereby theoptical spectrum. One modular adaptive optics unit embodiment of theadaptive optics scanning system includes the case where dispersioncompensating elements or systems may be used to compensate fordispersion in the light beam caused by optical material that was “seen”by the beam before the dispersion compensating elements or systems, orto pre-compensate for dispersion in the light beam caused by opticalmaterial that would be “seen” by the beam after the dispersioncompensating elements or systems. One modular adaptive optics unitembodiment includes the case where the dispersion compensating elementsor systems may include, but not be limited to, multilayer dielectricmirrors, optical prisms, diffractive optical elements, holographicoptical elements, liquid crystal optical elements, programmablediffractive optical elements, programmable pulse shapers, either asindependent dispersion compensating elements or in combination toachieve a desired amount of dispersion compensation.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.Furthermore, the foregoing describes the invention in terms ofembodiments foreseen by the inventor for which an enabling descriptionwas available, notwithstanding that insubstantial modifications of theinvention, not presently foreseen, may nonetheless represent equivalentsthereto.

What is claimed is:
 1. A method for scanning a sample, the methodcomprising: generating a first light from an emission source; directingthe first light to one or more adaptive optics element(s), the adaptiveoptics element(s) affecting the wavefront, affecting the intensity, oraffecting both the wavefront and intensity of the first light;controlling an angle and position of the first light with four or moreaxes of motion to preferentially interface the adaptive optics elementby creating or accommodating a beam pivot point at or near the adaptiveoptics element(s) while scanning the first light across the sample;controlling motion trajectories of the four or more axes of motion;conditioning and directing the first light to the sample; and measuringa second light from the sample.
 2. The method of claim 1, furthercomprising performing imaging of the sample.
 3. The method of claim 1,further comprising performing optical coherence tomography (OCT).
 4. Themethod of claim 1, further comprising generating the first light with alaser.
 5. The method of claim 1, further comprising: measuring anaberration in a third light from the sample or a point source within thesample; and determining an appropriate adaptive optics correction byusing information about the aberration.
 6. The method of claim 1,further comprising determining an appropriate adaptive optics correctionby using a wavefront sensorless adaptive optics optimization method. 7.The method of claim 1, wherein controlling an angle and position of thefirst light is performed using four galvanometer driven mirrors.
 8. Themethod of claim 1, wherein controlling an angle and position of thefirst light is performed using two fast steering mirrors.
 9. The methodof claim1, wherein controlling an angle and position of the first lightis performed using at least one MEMS mirror.
 10. The method of claim1,further comprising switching between at least a first and a secondemission sources using at least one axis of the four or more axes ofmotion.
 11. The method of claim 10, further comprising performing OCTimaging with the first emission source and microscopy imaging with thesecond emission source.
 12. The method of claim 1, further comprisingperforming different imaging modalities by switching between emissionsources.
 13. A method for scanning a sample, the method comprising:generating a first light from an emission source; directing the firstlight to one or more adaptive optics element(s), the adaptive opticselement(s) affecting the wavefront, affecting the intensity, oraffecting both the wavefront and intensity of the first light, whereinthe adaptive optics element(s) is (are) conjugated to a plane(s) outsideof the pupil plane to improve adaptive optics correction of aberrationsassociated with the sample; controlling an angle and position of thefirst light with two or more axis of motion to preferentially interfacethe adaptive optics element by creating or accommodating a beam pivotpoint at or near the adaptive optics element(s) while scanning the firstlight across the sample, wherein a light beam center of the first lightmoves on the adaptive optics elements(s) surface; controlling motiontrajectories of the two or more axes of motion; conditioning anddirecting the first light to the sample; and measuring a second lightfrom the sample.
 14. The method of claim 13, wherein at least oneadaptive optics element is conjugated to a plane that is at or near asource of aberration.
 15. An adaptive optics scanning system comprising:an emission source for generating a first light, the first light beingdirected through the adaptive optics scanning system to a sample; one ormore adaptive optics element(s), the adaptive optics element(s)affecting the wavefront, affecting the intensity, or affecting both thewavefront and intensity of the first light, wherein the adaptive opticselement(s) is (are) conjugated to a plane(s) outside of the pupil planeto improve adaptive optics correction of aberrations associated with thesample; an optical subsystem, the optical subsystem operating with twoor more axes of motion and controlling an angle and position of thefirst light to preferentially interface the adaptive optics element bycreating or accommodating a beam pivot point at or near the adaptiveoptics element(s) while scanning the first light across the sample,wherein a light beam center of the first light moves on the adaptiveoptics element(s) surface; a controller for controlling motiontrajectories of the two or more axes in the optical subsystem; sampledelivery optics, the sample delivery optics appropriately conditioningand directing the first light to the sample; and one or moredetector(s), the detector(s) measuring a second light from the sample.16. The adaptive optics scanning system of claim 15, wherein at leastone adaptive optics element is conjugated to a plane that is at orsubstantially near a source of aberration.
 17. The adaptive opticsscanning system of claim 15, wherein the optical subsystem comprises atleast one 4f relay.
 18. The method of claim 13, further comprisingdetermining an appropriate adaptive optics correction by using awavefront sensorless adaptive optics optimization method.
 19. Theadaptive optics scanning system of claim 15, wherein the sample deliveryoptics comprise a microscope objective and wherein the adaptive opticsscanning system performs laser scanning microscopy.
 20. The adaptiveoptics scanning system of claim 15, further comprising aninterferometer, wherein the adaptive optics scanning system performsoptical coherence tomography.